Antigen Retrieval Protocols

Antigen Retrieval Protocols http://www.ihcworld.com/epitope_retrieval.htm

The demonstration of many antigens can be significantly improved by the pretreatment with the antigen retrieval reagents that break the protein cross-links formed by formalin fixation and thereby uncover hidden antigenic sites. Antigen Retrieval Overview  Overview of Heat-induced Epitope Retrieval Techniques and Devices Room Temperature Epitope Retrieval (RTER)  Hydrochloric Acid Method (pH 1)  Formic Acid Method (pH 2) Heat Induced Epitope Retrieval (HIER)  Citrate Buffer Method (pH 6)  Citrate-EDTA Buffer Method (pH 6.2)  EDTA Method (pH 8)  Tris-EDTA Method (pH 9)  TBS Method (pH 9)  Tris Buffer Method (pH 10) Proteolytic Induced Epitope Retrieval (PIER)  Proteinase K Method  Trypsin Method  Pepsin Method  Pronase Method  Protease Method Frozen Section Epitope Retrieval  SDS Method  Heating En Bloc Method Free Floating Section Epitope Retrieval  Antigen Retrieval for Free Floating Sections Universal Antigen Retrieval Method  Citraconic Anhydride Method More antigen retrieval techniques and protocols

Overview of Heat-Induced Epitope Retrieval (HIER) Techniques and Devices Joe Myers, M.S., CT(ASCP) [email protected]

Loosely defined, heat-induced epitope retrieval (HIER) is a pre-treatment procedure often used prior to immunohistochemistry (IHC) or in situ hybridization (ISH) procedures to improve staining by modifying the molecular conformation of ‘target’ proteins through an exposure of slide-mounted specimen material (sectioned tissue and other cellular preparations) to a heated buffer solution. This modification process is necessary because, although aldehyde-based fixatives are excellent for preserving cellular morphology, they also cause protein cross-linking, resulting in the inability of some protein epitopes to bind complementary antibodies. HIER is commonly used in conjunction with enzyme digestion as a means of improving the reactivity of various antigens within IHC/ISH staining reactions. Since a variety of techniques and devices have been used to perform HIER, this review may be useful to individuals interested in identifying the most ideal method or instrument for their laboratory, and assist them in making better informed decisions. While a number of papers describing the advantages and disadvantages of various HIER methods for specific applications have been published (1,2), few have compared the technical differences between methods (3), or objectively presented the capabilities and shortcomings of existing HIER devices. HIER, also known simply as antigen retrieval (AR), was pioneered in the early 1990’s (4,5) after it had become apparent that use of enzyme digestion alone to improve IHC staining was inadequate. Although the expression “antigen retrieval” has gained widespread acceptance within the scientific community, alternative terms such as “protein unmasking”, “de-cloaking”, and “epitope recovery” have also been used to describe these procedures. As a general rule, all HIER procedures involve heating slide-mounted specimen material in a buffer solution, followed by a cooling-off period. Heat causes cross-linked protein epitopes to ‘unfold’ (in manner similar to DNA denaturation), while buffer solutions aid in maintaining the conformation of the unfolded protein. The primary differences between various HIER methods are the means by which such solutions are heated and exposed or applied to slides. During the early development of HIER, the most commonly employed methods for heating HIER solutions involved use of water baths, microwave ovens, and autoclaves. By the mid-1990’s, however, scientists began to experiment with ‘kitchen-grade’ vegetable steamers and pressure cookers as heat sources, finding that equally good results could be obtained with these devices. Recognizing that household appliances are limited in their ability to allow for protocol ‘customization’, development efforts in recent years have resulted in several devices designed specifically for performing HIER procedures in a laboratory environment. In their simplest form, most HIER devices consist of a primary metal or plastic chamber (into which secondary reagent containers are placed), surrounded by a reliable mechanism for heating the liquid in the secondary containers. The only limit to the number of slides (secondary reagent containers) that such a device can hold is the size of the primary chamber. Considering the importance of producing consistent results, the ideal HIER device: A) incorporates a precision-controlled heat o source, capable of maintaining temperatures at or above 100 C; B) holds a reasonable volume of retrieval buffer and slides; and C) minimizes the potential for evaporation and boiling of the HIER solution. These latter requirements are important because excessive evaporation: A) causes the salt concentration of the buffer to fluctuate; B) boiling can cause specimen material to be released from the slide; and C) boil-over can lead to exposure of ‘raw’ specimen material to the atmosphere, resulting in inadequate retrieval and less than ideal morphology, due to drying artefact. As a function of the inverse relationship between temperature and exposure time, devices that operate above o 100 C and prevent boiling (e.g. pressure cookers) have become very popular because they produce good results in the shortest possible timeframe. Other important aspects of HIER devices include the ease with which time and temperature settings can be adjusted, the amount of time needed to complete a ‘run’, the amount of operator intervention that is required, and the potential for the operator to be scalded while handling the device or the retrieval solutions. Ideally, HIER devices (or the protocols in which they are employed) should also provide a means of documenting performance, which ensures consistency and helps in meeting

various regulatory agency-imposed ‘QC’ standards. Table 1 provides a summary of the important characteristics of several common HIER methods.

Table1. HIER Method Comparison Comparative Characteristic

Water Bath

Temperature Range

25 to 100 C

85 to 95 C

50 to 100 C+

25 to 125 C

Heat-source Regulation

Good

Poor

Good

Excellent

Potential for Reagent Evaporation

Significant

Significant

Significant

Minimal

Potential for Boil-over

None

Minimal

Significant

Minimal

Potential to Scald Operator

Minimal

Moderate

Moderate

Significant

Maximum Slide Capacity per ‘Run’

(Variable)

72**

96**

96**

Maximum Reagent Capacity

(Variable)

750 ml***

1 litre***

1 litre***

o

Vegetable Steamer Microwave Oven o

o

Pressure Cooker* o

Note: All devices specified above are capable of holding both Coplin jars and TissueTek™-style slide racks/reagent containers *Refers to programmable devices, not ‘standard’ household appliances; see Device Comparison table for more information **Based on use of TissueTek™-style slide racks and reagent containers to hold retrieval solutions; although some models may hold more slides/containers than specified above, treatment of additional more slides may compromise performance characteristics (i.e. time, staining consistency) ***Based on use of TissueTek™-style slide racks/containers; use of Coplin jars results in a reduction in slide capacity and reagent ‘consumption’ Note: Tissue-Tek™ products are manufactured by Sakura Finetek

Recognizing that laboratorians needed to use reliable, inexpensive heat sources, scientists have employed a number of readily available devices, such as laboratory-grade water baths, autoclaves, and certain household appliances. Although simple in design and operation, the primary drawback of o water baths is the inability to achieve retrieval temperatures above 100 C (resulting in longer protocols), the requirement for manual filling and draining of buffers (requiring significant ‘hands-on’ time’), and the potential for retrieval solutions to evaporate during extended, high-temperature protocols. While the household-grade microwave oven also holds a prominent place in the field of HIER, it too has several shortcomings, most notably the difficulty in regulating temperature, the likelihood of boiling, and the fact that these units ‘loose power’ over time. Vegetable steamers, like water baths, o are often incapable of heating solutions above 95 C, resulting in exceptionally long protocols. In addition, vegetable steamers do not hold very many slides, relative to other devices. Pressure cookers have become very popular, primarily because of their higher operating temperature and ‘closed system’ design, which allows for shorter protocols and eliminates the potential for boiling. This is especially true of units that are retrofitted with a digital control panel and pressure gauge, since these modifications provide for better temperature regulation and permit the operator to monitor performance during the procedure. The important technical aspects of these devices can be found in Table 2.

Table 2. HIER Device Comparison Pickcell Laboratories

BioGenex Laboratories

Manufacturer

Biocare Medical

Vendor’s Web Address

www.biocare.net

COMPARATIVE CHARACTERISTICS

Decloaking Chamber™

The Retriever™

EZ Retriever™

PT Module™

Heat Source / Adjunctive Technology

Thermoelectric/ Pressure

Thermoelectric/ Pressure

Microwave

Thermoelectric

Reaction Chamber Material

Polypropylene*

Polypropylene*

Teflon®

Stainless Steel

Requires Proprietary Accessories

No

Yes (Slide Chambers)

No

Yes (Slide Racks)

Filling and Draining Method

Manual

Manual

Manual

Manual

Operating Temperature Range

25 to 100oC

120oC (Fixed)

25 to 107oC

60 to 102oC

antigenretriever.com www.biogenex.com

Lab Vision www.labvision.com

Intended/Optimal Operating Temperature

125oC

120oC

100oC

100oC

Processing Time (Range)

30 Seconds to 99 Hours

N/A (Fixed)

30 Seconds to 99 Hours

60 Minutes to 20 Hours

Intended/Optimal Processing Time

40 Minutes

2 Hours, 15 Minutes

40 minutes

80 minutes

Maximum Number of ‘Reaction Chambers’

4*

6*

4*

2*

Maximum Number of Slides Per Chamber

24*

18*

24*

24*

Maximum Number of Slides Per Run

96*

108*

96*

48*

Recommended Reagent Vol. Per Chamber

250 mL*

70 mL*

225 mL

1,500 mL

Maximum Reagent Volume Per Run

1 Liter*

420 mL*

900 mL

3.0 Liters**

Reagent Consumed Per Slide***

10.4 mL

3.9 mL

3.9 mL

62.5 mL

Capability of Storing Multiple Programs

No

No

No

No

Ability to Hold TissueTek™ Slide Racks

Yes

No

Yes

Yes

Ability to Hold Autostainer™ Slide Racks

No

No

No

Yes

*Based on use of TissueTek™-style slide racks and reagent containers (or similar, proprietary products) to hold retrieval solutions **Based on use of Autostainer™ slide racks ***Refers to amount of reagent consumed during an individual ‘run’, within applicable ‘reaction chambers’ Note: Autostainer™ systems are manufactured by Lab vision Corporation

Despite the fact that household appliances are commonly used within the laboratory environment, scientists agree that more standardization and specially-designed instruments are needed. It is interesting to note that one prominent IHC vendor seems to have had some difficulty deciding which ‘technology’ provides the best HIER results. A few years ago, they advocated use of a vegetable steamer, then began recommending use of a laboratory-grade water bath (possibly because of its application in the validation studies used to secure FDA approval for one of their proprietary IHC test kits); at present, they advocate use of and market a digitally-controlled pressure cooker. After more than ten years without the availability of a true lab instrument to perform these procedures, there are now several commercially available devices designed specifically for HIER (See Table 2). For example, PickCell Laboratories’ has introduced the 2100 Retriever™, which is a pressure cooker-like device (without the pressure gauge and time/temperature display found on similar units). The EZ Retriever™, from BioGenex Laboratories, is essentially an industrial-grade microwave oven containing four Teflon® reaction chambers and a temperature-monitoring probe. Like the devices upon which their operations are based (i.e. pressure cookers and microwave ovens), these new instruments possess many of the same shortcomings. At present, the only device that can easily be classified as laboratory instrument is the PT (PreTreatment) Module™ from Lab Vision Corporation. This semi-automated instrument was designed primarily for use with Lab Vision’s own Autostainer™ slide racks, which allows slides to be transferred directly from the PT Module™ to an Autostainer™ at the completion of the HIER procedure. The primary shortcomings of this device are that it requires the operator to fill and drain reagents from two large, removable stainless steel ‘tanks’, and consumes a disproportionately large volume of retrieval solution. No discussion of HIER methods and devices would be complete without mentioning the ability of some automated IHC slide staining systems to perform HIER. At present, there are more than eight different IHC stainers on the market, of which only three offer ‘on-board’ HIER capability. Unlike the devices described above, in which many slides are submerged in a heated buffer solution at the same time, HIER performed within IHC stainers usually involves processing slides individually. For example, Ventana Medical Systems’s Benchmark® stainer essentially ‘sprays’ HIER solution unto individual, horizontally-oriented slides within a rotary ‘carousel’, then heat each slide to a preset temperature, and rotate the carousel in order to repeat this process on other slides. In most protocols, the HIER solution is applied as many as nine times before HIER is considered completed. In a similar fashion, Vision-Biosystems’ Bond-maX™ stainer dispenses HIER reagents

onto rows of horizontally-arranged slides via a pipettor mechanism, and then whole rows are heated together. As interesting as these features are, the addition of HIER capability to an IHC slide stainer does nothing but increase the likelihood of specimen loss and drive up the cost of performing this otherwise ‘inexpensive’ procedure. The former problem is especially significant because the operator is usually not aware of specimen loss until an entire (expensive) IHC run has been completed, which usually requires repeating the IHC stain; the latter concern is discussed at greater length in the following section. Another important aspect of HIER that deserves discussion is the cost-effectiveness of various methods and devices. This is especially true when one considers that addition of HIER, or deparaffinization and HIER, to an automated protocol can substantially increase the overall cost of IHC (7). One important characteristic of HIER devices is the amount of reagent that is consumed during a ‘run’ or processing batch (shown in Table 2), and there are significant differences between devices. For example, previously-published data (8) have demonstrated that the cost of performing HIER on an automated IHC stainer can be as much as six times greater than performing HIER in a modified pressure cooker. Another example of a major difference in cost between two vendor’s methods and devices is shown in Table 3. Heat-induced epitope retrieval is a rather simple laboratory technique that has become an essential part of many IHC staining procedures. Although there a only a few basic methods, there are variety of devices used for HIER, ranging from modified household appliances to semi-automated laboratory instruments. Unfortunately, these variations also mean that HIER lacks standardization and cost controls. The purpose of this review, then, is to educate the buying public on the technical and financial aspects of HIER, with the hope that this information can be used to make better method selection and device acquisition decisions. References 1. Taylor CR, Shi SR, et al: Comparative study of antigen retrieval heating methods: Microwave, microwave and pressure cooker, autoclave, and steamer. Biotech Histochem 71(5): 263-70, 1996. 2. Pileri SA, Roncador G, et al: Antigen retrieval techniques in immunohistochemistry: A comparison of different methods. J Pathol 183(1): 116-23, 1997. 3. Tacha D and Teixeira M: History and overview of antigen retrieval: Methodologies and critical aspects. J Histotechnol 25(4): 237-42, 2002. 4. Shin RW, Iwaki T, et al: Hydrated autoclave pretreatment enhances Tau immunoreactivity in formalin-fixed normal and Alzheimer’s disease brain tissues. Lab Invest 64(5):693-702, 1991. 5. Shi S-R, Key ME and Kalra KL: Antigen retrieval in formalin-fixed, paraffin-embedded tissues: An enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39(6):741-48, 1991. 6. Myers J: Automated slide stainers for special stains, immunohistochemistry and in situ hybridization: A review. Med Lab Observer 36(1): 28-30, 2004. 7. Myers J: Reducing Immunohistochemistry Expense – Part 1. Advance for Administrators of the Laboratory 13 (9):16-18, 2004. 8. Myers J: Reducing Immunohistochemistry Expense – Part 2. Advance for Administrators of the Laboratory 13 (10):18-22, 2004.

Hydrochloric Acid (HCl) Antigen Retrieval Protocol Description: Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. The HCl based solution is designed to break the protein cross-links, therefore unmask the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections, thus enhancing staining intensity of antibodies. Solutions and Reagents: Hydrochloric Acid (HCl) Solution (2N in Distilled Water, pH 0.6-0.9): 10N (concentrated) HCl ----------------- 20 ml Distilled water ---------------------------- 80 ml Mix well and pH should be around 0.6-0.9 Incubate sections for 10-20 minutes at room temperature. Procedure: 1. De-paraffinize sections in 2 changes of xylene, 5 minutes each. 2. Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each. Then rinse in distilled water. 3. Incubate sections with 2N HCl solution for 10-20 minutes (optimal incubation time should be determined by user). 4. Rinse sections in PBS Tween 20 for 2x2 min. 5. Block sections with for 30 minutes. 6. Perform avidin/biotin blocking if necessary. 7. Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 C. 8. Rinse sections with PBS Tween 20 for 2x2 min. 9. Block sections with peroxidase blocking solution for 10 minutes. 10. Rinse with PBS Tween 20 for 3x2 min. 11. Proceed to standard immunohistochemistry protocol. Note: This method works well with BrdU and beta amyloid antibody staining.

Formic Acid Antigen Retrieval Protocol Description: Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. The formic acid based solution is designed to break the protein crosslinks, therefore unmask the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections, thus enhancing staining intensity of antibodies. Solutions and Reagents: Formic Acid Solution (10% in Distilled Water, pH1.6-2.0): 99% Formic acid ------------------------ 10 ml Distilled water -------------------------- 90 ml Mix well and pH should be around 1.6-2.0 Store at room temperature. Procedure: 1. De-paraffinize sections in 2 changes of xylene, 5 minutes each. 2. Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each. Then rinse in distilled water. 3. Incubate sections with formic acid solution for 10-20 minutes (optimal incubation time should be determined by user). 4. Rinse sections in PBS Tween 20 for 2x2 min. 5. Block sections with for 30 minutes. 6. Perform avidin/biotin blocking if necessary. 7. Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 C. 8. Rinse sections with PBS Tween 20 for 2x2 min. 9. Block sections with peroxidase blocking solution for 10 minutes. 10. Rinse with PBS Tween 20 for 3x2 min. 11. Proceed to standard immunohistochemistry protocol. Note: This method works well with beta amyloid antibodies staining. References: Kitamoto T, Ogomori K, Tateishi J, Prusiner SB (1987) Formic acid pretreatment enhances immunostaining of cerebral and systemic amyloids. Lab Invest. 57(2):230-6. PubMed Abstract

Citrate Buffer Antigen Retrieval Protocol Description: Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. The citrate based solution is designed to break the protein cross-links, therefore unmask the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections, thus enhancing staining intensity of antibodies. Solutions and Reagents: Sodium Citrate Buffer (10mM Sodium Citrate, 0.05% Tween 20, pH 6.0): Tri-sodium citrate (dihydrate) --------- 2.94 g Distilled water --------------------------- 1000 ml Mix to dissolve. Adjust pH to 6.0 with 1N HCl and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4 C for longer storage. Note: this buffer is commonly used and works perfectly with many antibodies. It gives very nice intense staining with very low background. Citrate Buffer (10mM Citric Acid, 0.05% Tween 20, pH 6.0): Citric acid (anhydrous) --------------- 1.92 g Distilled water -------------------------- 1000 ml Mix to dissolve. Adjust pH to 6.0 with 1N NaOH and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4 C for longer storage. Procedure: 1. De-paraffinize sections in 2 changes of xylene, 5 minutes each. 2. Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each. Then rinse in distilled water. 3. Pre-heat steamer or water bath with staining dish containing Sodium Citrate Buffer or Citrate Buffer until temperature reaches 95-100 C. 4. Immerse slides in the staining dish. Place the lid loosely on the staining dish and incubate for 20-40 minutes (optimal incubation time should be determined by user). 5. Turn off steamer or water bath and remove the staining dish to room temperature and allow the slides to cool for 20 minutes. 6. Rinse sections in PBS Tween 20 for 2x2 min. 7. Block sections with for 30 minutes. 8. Perform avidin/biotin blocking if necessary. 9. Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 C. 10. Rinse sections with PBS Tween 20 for 2x2 min. 11. Block sections with peroxidase blocking solution for 10 minutes. 12. Rinse with PBS Tween 20 for 3x2 min. 13. Proceed to standard immunohistochemistry protocol. Note: Microwave, pressure cooker and autoclave can be used as alternative heating source to replace steamer or water bath. References: 1. Shi SR, Chaiwun B, Young L, Cote RJ, Taylor CR. Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J Histochem Cytochem. 1993 Nov;41(11):1599-604. PubMed Abstract 2. Kanai K, Nunoya T, Shibuya K, Nakamura T, Tajima M (1998) Variations in effectiveness of antigen retrieval pretreatments for diagnostic immunohistochemistry. Res Vet Sci. 64(1):57-61. PubMed Abstract 3. Brown RW, Chirala R (1995) Utility of microwave-citrate antigen retrieval in diagnostic immunohistochemistry. Mod Pathol. 8(5):515-20. PubMed Abstract 4. Morgan JM, Navabi H, Schmid KW, Jasani B. Possible role of tissue-bound calcium ions in citrate-mediated hightemperature antigen retrieval. J Pathol. 1994 Dec;174(4):301-7. PubMed Abstract 5. Pellicer EM, Sundblad A (1994) Antigen retrieval by microwave oven with buffer of citric acid. Medicina (B Aires). 54(2):12932. PubMed Abstract 6. Shi SR, Chaiwun B, Young L, Cote RJ, Taylor CR (1993) Antigen retrieval technique utilizing citrate buffer or urea solution for immunohistochemical demonstration of androgen receptor in formalin-fixed paraffin sections. J Histochem Cytochem. 41(11):1599-604. PubMed Abstract

Citrate-EDTA Antigen Retrieval Protocol Description: Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. The citrate-EDTA based solution is designed to break the protein crosslinks, therefore unmask the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections, thus enhancing staining intensity of antibodies. Solutions and Reagents: Citrate-EDTA Buffer (10mM Citric Acid, 2mM EDTA, 0.05% Tween 20, pH 6.2): Citric acid (anhydrous) ----------------- 1.92 g EDTA (Sigma, Cat# E-5134) ------------ 0.74 g Distilled water --------------------------- 1000 ml Mix to dissolve. Adjust pH to 6.2 and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4 C for longer storage. Procedure: 1. De-paraffinize sections in 2 changes of xylene, 5 minutes each. 2. Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each. Then rinse in distilled water. 3. Pre-heat steamer or water bath with staining dish containing Citrate-EDTA buffer until temperature reaches 95-100 C. 4. Immerse slides in the staining dish. Place the lid loosely on the staining dish and incubate for 20-40 minutes (optimal incubation time should be determined by user). 5. Turn off steamer or water bath and remove the staining dish to room temperature and allow the slides to cool for 20 minutes. 6. Rinse sections in PBS Tween 20 for 2x2 min. 7. Block sections with for 30 minutes. 8. Perform avidin/biotin blocking if necessary. 9. Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 C. 10. Rinse sections with PBS Tween 20 for 2x2 min. 11. Block sections with peroxidase blocking solution for 10 minutes. 12. Rinse with PBS Tween 20 for 3x2 min. 13. Proceed to standard immunohistochemistry protocol. Note: Microwave, pressure cooker or autoclave can be used as alternative heating source to replace steamer or water bath. References: 1. Sheriffs IN, Rampling D, Smith VV (2001) Paraffin wax embedded muscle is suitable for the diagnosis of muscular dystrophy. J Clin Pathol. 54(7):517-20. PubMed Abstract

EDTA Buffer Antigen Retrieval Protocol Description: Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. The EDTA based solution is designed to break the protein cross-links, therefore unmask the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections, thus enhancing staining intensity of antibodies. Solutions and Reagents: EDTA Buffer (1mM EDTA, 0.05% Tween 20, pH 8.0): EDTA (Sigma, Cat# E-5134) ----------- 0.37 g Distilled water -------------------------- 1000 ml Mix to dissolve. Adjust pH to 8.0 using 1N NaOH. Then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4 C for longer storage. Note: This buffer works excellent for many antibodies, but it often gives high background staining (maybe due to endogenous biotin revealed after this pretreatment). So primary antibody can often be highly diluted. It is very useful for low affinity antibodies or when tissue antigens are not intense. Procedure: 1. De-paraffinize sections in 2 changes of xylene, 5 minutes each. 2. Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each. Then rinse in distilled water. 3. Pre-heat steamer or water bath with staining dish containing EDTA Buffer until temperature reaches 95-100 C. 4. Immerse slides in the staining dish. Place the lid loosely on the staining dish and incubate for 20-40 minutes (optimal incubation time should be determined by user). 5. Turn off steamer or water bath and remove the staining dish to room temperature and allow the slides to cool for 20 minutes. 6. Rinse sections in PBS Tween 20 for 2x2 min. 7. Block sections with for 30 minutes. 8. Perform avidin/biotin blocking if necessary. 9. Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 C. 10. Rinse sections with PBS Tween 20 for 2x2 min. 11. Block sections with peroxidase blocking solution for 10 minutes. 12. Rinse with PBS Tween 20 for 3x2 min. 13. Proceed to standard immunohistochemistry protocol. Note: Microwave, pressure cooker or autoclave can be used as alternative heating source to replace steamer or water bath. References: 1. Pileri SA, Roncador G, Ceccarelli C, et al (1997) Antigen retrieval techniques in immunohistochemistry: comparison of different methods. J Pathol. 183(1):116-23. PubMed Abstract 2. Morgan JM, Navabi H, Schmid KW, Jasani B (1994) Possible role of tissue-bound calcium ions in citrate-mediated hightemperature antigen retrieval. J Pathol. 174(4):301-7. PubMed Abstract

Tris-EDTA Buffer Antigen Retrieval Protocol Description: Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. The Tris-EDTA based solution is designed to break the protein crosslinks, therefore unmask the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections, thus enhancing staining intensity of antibodies. Solutions and Reagents: Tris-EDTA Buffer (10mM Tris Base, 1mM EDTA Solution, 0.05% Tween 20, pH 9.0): Tris Base -------------------------------- 1.21 g EDTA ------------------------------------- 0.37 g Distilled water -------------------------- 1000 ml (100 ml to make 10x, 50 ml to make 20x) Mix to dissolve. pH is usually at 9.0 and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4 C for longer storage. Note: This buffer works excellent for many antibodies, but it often gives high background staining (maybe due to endogenous biotin revealed after this pretreatment). So primary antibody can often be highly diluted. It is very useful for low affinity antibodies or when tissue antigens are not intense. Procedure: 1. De-paraffinize sections in 2 changes of xylene, 5 minutes each. 2. Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each. Then rinse in distilled water. 3. Pre-heat steamer or water bath with staining dish containing Tris-EDTA Buffer until temperature reaches 95-100 C. 4. Immerse slides in the staining dish. Place the lid loosely on the staining dish and incubate for 20-40 minutes (optimal incubation time should be determined by user). 5. Turn off steamer or water bath and remove the staining dish to room temperature and allow the slides to cool for 20 minutes. 6. Rinse sections in PBS Tween 20 for 2x2 min. 7. Block sections with for 30 minutes. 8. Perform avidin/biotin blocking if necessary. 9. Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 C. 10. Rinse sections with PBS Tween 20 for 2x2 min. 11. Block sections with peroxidase blocking solution for 10 minutes. 12. Rinse with PBS Tween 20 for 3x2 min. 13. Proceed to standard immunohistochemistry protocol. Note: Microwave, pressure cooker or autoclave can be used as alternative heating source to replace steamer or water bath.

Tris Buffered Saline (TBS) Antigen Retrieval Protocol Description: Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. The Tris based solution is designed to break the protein cross-links, therefore unmask the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections, thus enhancing staining intensity of antibodies. Solutions and Reagents: Tris Buffered Saline (TBS) (0.05M TBS, 0.05% Tween 20, pH 9.0): Tris Base --------------------------------- 6.1 g Sodium Chloride ------------------------- 8.8 g Distilled water --------------------------- 1000 ml Mix to dissolve. Adjust pH to 9.0 using concentrated HCl and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4 ºC for longer storage. Procedure: 1. De-paraffinize sections in 2 changes of xylene, 5 minutes each. 2. Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each. Then rinse in distilled water. 3. Pre-heat steamer or water bath with staining dish containing TBS until temperature reaches 95-100 C. 4. Immerse slides in the staining dish. Place the lid loosely on the staining dish and incubate for 20-40 minutes (optimal incubation time should be determined by user). 5. Turn off steamer or water bath and remove the staining dish to room temperature and allow the slides to cool for 20 minutes. 6. Rinse sections in PBS Tween 20 for 2x2 min. 7. Block sections with for 30 minutes. 8. Perform avidin/biotin blocking if necessary. 9. Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 C. 10. Rinse sections with PBS Tween 20 for 2x2 min. 11. Block sections with peroxidase blocking solution for 10 minutes. 12. Rinse with PBS Tween 20 for 3x2 min. 13. Proceed to standard immunohistochemistry protocol. Note: Microwave, pressure cooker or autoclave can be used as alternative heating source to replace steamer or water bath. References: 1. Taylor CR, Shi SR, Chen C, Young L, Yang C, Cote RJ (1996) Comparative study of antigen retrieval heating methods: microwave, microwave and pressure cooker, autoclave, and steamer. Biotech Histochem. 71(5):263-70. PubMed Abstract 2. Evers P, Uylings HB (1997) An optimal antigen retrieval method suitable for different antibodies on human brain tissue stored for several years in formaldehyde fixative. J Neurosci Methods. 72(2):197-207. PubMed Abstract

Tris Buffer Antigen Retrieval Protocol Description: Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. The Tris based solution is designed to break the protein cross-links, therefore unmask the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections, thus enhancing staining intensity of antibodies. Solutions and Reagents: Tris-Buffer (10mM Tris Base, 0.05% Tween 20, pH 10): Tris Base -------------------------------- 1.21 g Distilled water -------------------------- 1000 ml Mix to dissolve. Adjust pH to 10 using 1N NaOH and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4 ºC for longer storage. Note: It is sometime occurred with tissue damage or sections detached from slides when using this method. It is probably due to high pH value. Procedure: 1. De-paraffinize sections in 2 changes of xylene, 5 minutes each. 2. Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each. Then rinse in distilled water. 3. Pre-heat steamer or water bath with staining dish containing Tris Buffer until temperature reaches 95-100 C. 4. Immerse slides in the staining dish. Place the lid loosely on the staining dish and incubate for 20-40 minutes (optimal incubation time should be determined by user). 5. Turn off steamer or water bath and remove the staining dish to room temperature and allow the slides to cool for 20 minutes. 6. Rinse sections in PBS Tween 20 for 2x2 min. 7. Block sections with for 30 minutes. 8. Perform avidin/biotin blocking if necessary. 9. Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 C. 10. Rinse sections with PBS Tween 20 for 2x2 min. 11. Block sections with peroxidase blocking solution for 10 minutes. 12. Rinse with PBS Tween 20 for 3x2 min. 13. Proceed to standard immunohistochemistry protocol. Note: Microwave, pressure cooker or autoclave can be used as alternative heating source to replace steamer or water bath.

References: 1. Taylor CR, Shi SR, Chen C, Young L, Yang C, Cote RJ (1996) Comparative study of antigen retrieval heating methods: microwave, microwave and pressure cooker, autoclave, and steamer. Biotech Histochem. 71(5):263-70. PubMed Abstract

Proteinase K Antigen Retrieval Protocol Description: Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. The proteinase K based solution is designed to break the protein crosslinks, therefore unmask the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections, thus enhancing staining intensity of antibodies. Solutions and Reagents:

Proteinase K Solution (Method 1) (20 ug/ml in TE Buffer, pH 8.0): TE Buffer (50mM Tris Base, 1mM EDTA, 0.5% Triton X-100, pH 8.0): Tris Base -------------------------------- 6.10 g EDTA ------------------------------------- 0.37 g Triton X-100 ---------------------------- 5 ml Distilled water ------------------------- 1000 ml Mix to dissolve. Adjust pH 8.0 using concentrated HCl (10N HCl). Store at room temperature. Proteinase K Stock Solution (20x, 400 ug/ml or 12 units/ml): Proteinase K (30 units/mg)----------- 0.008 g (8 mg) TE Buffer, pH8.0 ---------------------- 10 ml Glycerol --------------------------------- 10 ml Add proteinase K to TE buffer until dissolved. Then add glycerol and mix well. Aliquot and store at –20 ºC for 2-3 years. Working Solution (1x, 20 ug/ml or 0.6 units/ml): Proteinase K Stock Solution (20x) ------ 1 ml TE Buffer, pH8.0 ------------------------- 19 ml Mix well. This solution is stable for 6 month at 4 ºC. Proteinase K Solution (Method 2) (20 ug/ml in TE-CaCl2 Buffer, pH 8.0): TE-CaCl2 Buffer (50mM Tris Base, 1mM EDTA, 5mM CaCl2, 0.5% Triton X-100, pH 8.0): Tris Base -------------------------------- 6.10 g EDTA ------------------------------------ 0.37 g CaCl2 ------------------------------------ 0.56 g Triton X-100 ---------------------------- 5 ml Distilled water ------------------------- 1000 ml Mix to dissolve. Adjust to pH 8.0 using concentrated HCl (10N HCl). Store this buffer at room temperature. Proteinase K Stock Solution (20x, 400 ug/ml or 12 units/ml): Proteinase K (30 units/mg) ----------- 0.008 g (8 mg) TE-CaCl2 Buffer, pH8.0 --------------- 10 ml Glycerol --------------------------------- 10 ml Add proteinase K to TE-CaCl2 buffer until dissolved. Then add glycerol and mix well. Aliquot and store at –20 ºC for 2-3 years. Working Solution (1x, 20 ug/ml or 0.6 units/ml): Proteinase K Stock Solution (20x) ------ 1 ml TE-CaCl2 Buffer, pH8.0 ------------------ 19 ml Mix well. This solution is stable for 6 month at 4 ºC. Procedure: 1. De-paraffinize sections in 2 changes of xylene, 5 minutes each. 2. Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each. 3. Rinse in distilled water. 4. Cover sections with Proteinase K working solution and incubate 10-20 minutes at 37 ºC in humidified chamber (optimal incubation time may vary depending on tissue type and degree of fixation, and should be determined by user). 5. Allow sections to cool at room temperature for 10 minutes. 6. Rinse sections in PBS Tween 20 for 2x2 min.

7. Block sections with for 30 minutes. 8. Perform avidin/biotin blocking if necessary. 9. Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 ºC. 10. Rinse sections with PBS Tween 20 for 2x2 min. 11. Block sections with peroxidase blocking solution for 10 minutes. 12. Rinse with PBS Tween 20 for 3x2 min. 13. Proceed to standard immunohistochemistry protocol. Notes: 1. This method tends to cause tissue damage for under-fixed tissues. Select appropriate incubation time (5-20 minutes) and temperature (20 ºC to 60 ºC) for a specific application and do not over-digest tissues especially for the under-fixed tissues. 2. Method 2 contains CaCl2, where the Ca increase enzyme activity.

2+

can activate proteinase K enzyme by 20-25%, therefore

References: 1. Ramos-Vara JA, Beissenherz ME. Optimization of immunohistochemical methods using two different antigen retrieval methods on formalin-fixed paraffin-embedded tissues: experience with 63 markers. J Vet Diagn Invest. 2000 Jul;12(4):307-11. PubMed Abstract 2. Proteinase K - Enzyme Explorer (Sigma) 3. Proteinase K Wikipedia Entry

Trypsin Antigen Retrieval Protocol Description: Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. The trypsin based solution is designed to break the protein cross-links, therefore unmask the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections, thus enhancing staining intensity of antibodies. Solutions and Reagents: Trypsin Solution: Trypsin Stock Solution (0.5% in Distilled Water): Trypsin -------------------------------- 50 mg DDistilled water ------------------------ 10 ml Mix to dissolve. Store at -20 ºC. Calcium Chloride Stock Solution (1%): CCalcium chloride --------------------- 0.1 g Distilled water ----------------------- 10 ml Mix well and store at 4 ºC. Trypsin Working Solution (0.05%): Trypsin stock solution (0.5%) ------------ 1 ml Calcium chloride stock solution 1% ------ 1 ml Distilled Water ----------------------------- 8 ml Adjust pH to 7.8 with 1N N NaOH. Store at 4 ºC for one month or -20 ºC for long term storage Procedure: 1. De-paraffinize sections in 2 changes of xylene, 5 minutes each. 2. Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each. 3. Rinse in distilled water. 4. Cover sections with trypsin working solution and incubate for 10-20 minutes at 37 C in humidified chamber (optimal incubation time may vary depending on tissue type and degree of fixation, and should be determined by user). 5. Allow sections to cool at room temperature for 10 minutes. 6. Rinse sections in PBS Tween 20 for 2x2 min. 7. Block sections with for 30 minutes. 8. Perform avidin/biotin blocking if necessary. 9. Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 C. 10. Rinse sections with PBS Tween 20 for 2x2 min. 11. Block sections with peroxidase blocking solution for 10 minutes. 12. Rinse with PBS Tween 20 for 3x2 min. 13. Proceed to standard immunohistochemistry protocol. Notes: This method tends to produce tissue damage so incubation time is import factor to consider when using this method. Select appropriate incubation time for a specific application. References: 1. Ashton-Key M, Jessup E, Isaacson PG (1996) Immunoglobulin light chain staining in paraffin-embedded tissue using a heat mediated epitope retrieval method. Histopathology. 29(6):525-31. PubMed Abstract 2. Kashima K, Yokoyama S, Daa T, Nakayama I, Nickerson PA, Noguchi S (1997) Cytoplasmic biotin-like activity interferes with immunohistochemical analysis of thyroid lesions: a comparison of antigen retrieval methods. Mod Pathol. 10(5):515-9. PubMed Abstract 3. Frost AR, Sparks D, Grizzle WE (2000) Methods of antigen recovery vary in their usefulness in unmasking specific antigens in immunohistochemistry. Appl Immunohistochem Mol Morphol. 8(3):236-43. PubMed Abstract

Pepsin Antigen Retrieval Protocol Description: Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. The pepsin based solution is designed to break the protein cross-links, therefore unmask the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections, thus enhancing staining intensity of antibodies.

Solutions and Reagents:

Pepsin Stock Solution (1% in 10mM HCl): Pepsin ------------------------------------- 100 mg 10mM HCl (pH 2.0) ---------------------- 10 ml Mix to dissolve. Store at -20 ºC Pepsin Working Solution (0.5% in 5mM HCl): Pepsin Stock Solution (0.5%) ------------ 1 ml Distilled water ---------------------------- 1 ml Mix well. Procedure: 1. De-paraffinize sections in 2 changes of xylene, 5 minutes each. 2. Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each. 3. Rinse in distilled water. 4. Cover sections with pepsin working solution and incubate for 10-20 minutes at 37 C in humidified chamber (optimal incubation time may vary depending on tissue type and degree of fixation, and should be determined by user). 5. Allow sections to cool at room temperature for 10 minutes. 6. Rinse sections in PBS Tween 20 for 2x2 min. 7. Block sections with for 30 minutes. 8. Perform avidin/biotin blocking if necessary. 9. Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 C. 10. Rinse sections with PBS Tween 20 for 2x2 min. 11. Block sections with peroxidase blocking solution for 10 minutes. 12. Rinse with PBS Tween 20 for 3x2 min. 13. Proceed to standard immunohistochemistry protocol. Note: This method tends to produce tissue damage so incubation time is import factor to consider when using this method. Select appropriate incubation time for a specific application.

Pronase Antigen Retrieval Protocol Description: Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. The pronase based solution is designed to break the protein cross-links, therefore unmask the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections, thus enhancing staining intensity of antibodies. Solutions and Reagents: Pronase Solution (0.05% in PBS): Pronase --------------------------------- 0.005 g PBS --------------------------------------- 10 ml Mix well and store at -20 C Pronase Solution (0.1% in PBS): Pronase --------------------------------- 0.01 g PBS --------------------------------------- 10 ml Mix well and store at -20 C Procedure: 1. De-paraffinize sections in 2 changes of xylene, 5 minutes each. 2. Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each. 3. Rinse in distilled water. 4. Cover sections with pronase working solution and incubate for 10-20 minutes at 37 C in humidified chamber (optimal incubation time may vary depending on tissue type and degree of fixation, and should be determined by user). 5. Allow sections to cool at room temperature for 10 minutes. 6. Rinse sections in PBS Tween 20 for 2x2 min. 7. Block sections with for 30 minutes. 8. Perform avidin/biotin blocking if necessary. 9. Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 C. 10. Rinse sections with PBS Tween 20 for 2x2 min. 11. Block sections with peroxidase blocking solution for 10 minutes. 12. Rinse with PBS Tween 20 for 3x2 min. 13. Proceed to standard immunohistochemistry protocol. Note: This method tends to pruduce tissue damage so pronase concentration and incubation time are import factors to consider when using this method. Select appropriate pronase concentration and incubation time for a specific application.

Protease Antigen Retrieval Protocol Description: Formalin or other aldehyde fixation forms protein cross-links that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. The protease based solution is designed to break the protein cross-links, therefore unmask the antigens and epitopes in formalin-fixed and paraffin embedded tissue sections, thus enhancing staining intensity of antibodies.

Solutions and Reagents:

Protease Solution (0.05% in Distilled Water) Protease ----------------------------------- 5 mg Distilled water --------------------------- 10 ml Mix to dissolve. Adjust to pH 7.8 using 1N NaOH. Store at -20 ºC.

Procedure: 1. De-paraffinize sections in 2 changes of xylene, 5 minutes each. 2. Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each. 3. Rinse in distilled water. 4. Cover sections with protease working solution and incubate for 10-20 minutes at 37 C in humidified chamber (optimal incubation time may vary depending on tissue type and degree of fixation, and should be determined by user). 5. Allow sections to cool at room temperature for 10 minutes. 6. Rinse sections in PBS Tween 20 for 2x2 min. 7. Block sections with for 30 minutes. 8. Perform avidin/biotin blocking if necessary. 9. Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 C. 10. Rinse sections with PBS Tween 20 for 2x2 min. 11. Block sections with peroxidase blocking solution for 10 minutes. 12. Rinse with PBS Tween 20 for 3x2 min. 13. Proceed to standard immunohistochemistry protocol. Notes: This method tends to produce tissue damage so incubation time is import factor to consider when using this method. Select appropriate incubation time for a specific application. References: Battifora H, Kopinski M (1986) The influence of protease digestion and duration of fixation on the immunostaining of keratins. A comparison of formalin and ethanol fixation. J Histochem Cytochem. 34(8):1095-100. PubMed Abstract

Antigen Retrieval Method for Cryostat Frozen Sections Description: This is a simple method for antigen retrieval on aldehyde-fixed cryostat tissue sections or cultured cells. In many case, a brief 5 minutes pretreatment with 1% sodium dodecyl sulfate (SDS) produced a dramatic increase in staining intensity by immunohistochemistry and immunofluorescence. Solutions and Reagents: 1% Sodium Dodecyl Sulfate (SDS) in PBS: SDS ---------------------------------- 1 g 0.01M PBS (pH 7.4) ---------------- 100 ml Mix to dissolve. Procedure: 1. Rinse sections three times for 5 min each in PBS. 2. Cover sections with 1% SDS solution and incubate for 5 minutes at room temperature. 3. Rinse sections three times for 5 min each in PBS. It is important to wash sections well, otherwise residual SDS will denature the antibodies subsequently applied to sections. 4. Incubate sections in serum blocking solution. 5. Incubate in the primary antibody and complete immunohistochemical staining steps as desired. References: Brown D, Lydon J, McLaughlin M, Stuart-Tilley A, Tyszkowski R, Alper S (1996) Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS). Histochem Cell Biol. 105(4):261-7. PubMed Abstract

Antigen Retrieval Method for Frozen Sections Description: Although AR by heating is widely effective, the majority of the existing methods are designed for paraffin-embedded sections. Frozen sections have been generally exempt from these methods because such sections are fragile and are easily destroyed by heating. A recent report demonstrates a simple alternative method for AR that can be used for aldehyde-fixed frozen sections. After fixation in paraformaldehyde, tissue blocks were heated in retrieval solutions and then frozen with dry ice. This method not only greatly enhanced the immunoreactivity for a wide range of antigens, especially for nuclear proteins, but also effectively lowered the background staining in some cases. Solutions and Reagents: Citrate Buffer (10mM Sodium Citrate Buffer, 0.05% Tween 20, pH 6.0): Tri-sodium citrate ---------------------- 2.94 g Distilled water -------------------------- 1000 ml Mix to dissolve. Adjust pH to 6.0 with 1N HCl and then add 0.5 ml of Tween 20 and mix well. Store this solution at room temperature for 3 months or at 4 C for longer storage. Procedure: 1. Prepare tissues fixed with 4% paraformaldehyde in 0.1M PB, pH 7.5. The tissue blocks should be cut to a proper size (e.g., slices 3–5 mm thick). 2. Immerse the tissue blocks in a retrieval solution (10 mM sodium citrate buffer, pH 6.0) at 4 ºC overnight. 3. Place the tissue blocks in a small, heat-resistant basket and immerse in boiling retrieval solution (200–500 ml) with gentle stirring using a hot plate for 3–5 min. For heating, a conventional burner can also be used. 4. Immediately place the tissue blocks in cold 30% sucrose in PBS and incubate at 4 ºC until the blocks sink. 5. Immerse the tissue blocks in an embedding medium and freeze quickly with crushed dry ice. The frozen tissue blocks can now be stored at -80 ºC. 6. Cut frozen sections with a cryostat and mount them on glass slides. Free-floating sections can also be used. 7. Dry the sections well for more than 1 day or several days at RT to prevent detachment of the sections during processing. 8. Proceed to standard immunohistochemistry procedure.

References: 1. Ino H (2003) Antigen retrieval by heating en bloc for pre-fixed frozen material. J Histochem Cytochem. 51(8):995-1003. PubMed Abstract 2. Evers P, Uylings HB (1994) Effects of microwave pretreatment on immunocytochemical staining of vibratome sections and tissue blocks of human cerebral cortex stored in formaldehyde fixative for long periods. J Neurosci Methods. 55(2):163-72. PubMed Abstract

Antigen Retrieval Method for Free Floating Sections Description: We describe here a simple, reliable and sensitive antigen retrieval method that uses water-bath heating. By this method, the temperature can be precisely controlled to yield effective antigen retrieval with minimal tissue damage in free-floating sections. We found that the best results were obtained with a 30 min incubation in a 10–50 mM sodium citrate solution (pH 8.5–9.0) preheated to and maintained at 80°C in a water-bath, followed by 30 min incubation in 0.3–3% non-fat dry milk to reduce non-specific staining. This method is highly effective for both 40 um free floating sections, slide-mounted cryostat sections and paraffin-embedded slide-mounted sections. Solutions and Reagents: Sodium Citrate Buffer (10mM Sodium Citrate Buffer, pH 8.5): Tri-sodium citrate ---------------------- 2.94 g Distilled water -------------------------- 1000 ml Mix to dissolve. Adjust pH to 8.5 and store this solution at room temperature for 3 months or at 4 C for longer storage. Procedure: 1. Rinse sections three times for 5 min each in 0.1 M PB (pH 7.4). 2. Transfer the sections to 10 mM sodium citrate buffer (pH 8.5) preheated to 80°C in water bath. 3. Maintain this temperature and keep sections in this solution for 30 min. 4. Retain the sections in this solution while allowing the solution to cool to room temperature. 5. Rinse the sections three times for 5 min each in 0.1 M PB (pH 7.4). 6. Immerse the sections in 2% non-fat dry milk in 0.1 M PB (pH 7.4) containing 0.3% Triton X100–0.01% sodium azide for 30–60 min. 7. Incubate in the primary antibody and complete immunohistochemical staining steps as desired. References: 1. Yun Jiao, et al (1999) A simple and sensitive antigen retrieval method for free-floating and slide-mounted tissue sections. Journal of Neuroscience Methods 93:149–162. 2. Evers P, Uylings HB (1994) Effects of microwave pretreatment on immunocytochemical staining of vibratome sections and tissue blocks of human cerebral cortex stored in formaldehyde fixative for long periods. J Neurosci Methods. 55(2):163-72. PubMed Abstract

Antigen Retrieval Protocols - Universal Antigen Retrieval Method Description: Formalin is a commonly used fixative for tissue preservation in histopathology labs. A major adverse effect of this fixative is the concealing of tissue antigens by protein cross-linking through formaldehyde reaction with the protein amino groups. Citraconylation by citraconic anhydride provides an easy method for the reversible blocking of protein amino groups. The method provides efficient AR for successful immunostaining of a wide variety of antigens under an optimized condition. Solutions and Reagents: 0.05% Citraconic Anhydride Buffer (pH 7.4): Citraconic anhydride ------------------ 0.5 g Distilled water --------------------- --- 1000 ml Mix to dissolve. Adjust pH to 7.4 Procedure: 1. De-paraffinize sections in 2 changes of xylene, 5 minutes each. 2. Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each. Then rinse in distilled water. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Pre-heat steamer or water bath with staining dish containing Citraconic Anhydride Buffer until temperature reaches 95-100 C. Immerse slides in the staining dish. Place the lid loosely on the staining dish and incubate for 20-40 minutes (optimal incubation time should be determined by user). Turn off steamer or water bath and remove the staining dish to room temperature and allow the slides to cool for 20 minutes. Rinse sections in PBS Tween 20 for 2x2 min. Block sections with for 30 minutes. Perform avidin/biotin blocking if necessary. Incubate sections with primary antibody at appropriate dilution in primary antibody dilution buffer for 1 hour at room temperature or overnight at 4 C. Rinse sections with PBS Tween 20 for 2x2 min. Block sections with peroxidase blocking solution for 10 minutes. Rinse with PBS Tween 20 for 3x2 min. Proceed to standard immunohistochemistry protocol.

Note: Since citraconic anhydride is considered a toxic agent, it is necessary to use a hood and to prevent contact with skin and eyes when performing the AR procedure, although the concentration needed was very low.

References: 1. Namimatsu S, Ghazizadeh M and Sugisaki Y (2005) Reversing the effects of formalin fixation with citraconic anhydride and heat: a universal antigen retrieval method. J Histochem Cytochem. 53(1):3-11. PubMed Abstract

Antigen Retrieval Methods & Techniques on Literature Overview:    

Antigen Retrieval Techniques: Current Perspectives Antigen Retrieval Immunohistochemistry: Past, Present, and Future Fixation and epitope retrieval in diagnostic immunohistochemistry: a concise review with practical considerations Antigen retrieval immunohistochemistry and molecular morphology in the year 2001

Mechanism:         

Role of calcium chelation in high-temperature antigen retrieval at different pH values Possible role of tissue-bound calcium ions in citrate-mediated high-temperature antigen retrieval Microwave-antigen retrieval: the importance of pH of the retrieval solution for MIB-1 staining Antigen retrieval in formaldehyde-fixed human brain tissue - pH, temp, duration of heating factors Application of the critical molar concentration concept to heat-mediated antigen retrieval in immunohistochemistry A molecular mechanism of formalin fixation and antigen retrieval DNA Extraction from Archival Formalin-fixed, Paraffin-embedded Tissue Sections Based on the Antigen Retrieval Principle: Heating Under the Influence of pH Heat-induced antigen retrieval restores electrostatic forces: prolonging the antibody incubation as an alternative Effects of different pH values of fixatives and antigen retrieval buffers on immunohistochemical staining

Methods Comparison:     

A comparison of microwave heating and proteolytic pretreatment antigen retrieval techniques in formalin fixed, paraffin embedded tissues Significance of Wet Autoclave Pretreatment in Immunohistochemistry A comparison of methods for heat-mediated antigen retrieval for immunoelectron microscopy: demonstration of cytokeratin No. 18 in normal and neoplastic hepatocytes High temperature antigen retrieval and loss of nuclear morphology: a comparison of microwave and autoclave techniques Comparative study of antigen retrieval heating methods: microwave, microwave and pressure cooker, autoclave, and steamer 

     

Wet autoclave pretreatment for antigen retrieval in diagnostic immunohistochemistry

Development of an optimal protocol for antigen retrieval: a 'test battery' approach exemplified with reference to the staining of retinoblastoma protein (pRB) in formalin-fixed paraffin sections Antigen retrieval techniques in immunohistochemistry: comparison of different methods Variations in effectiveness of antigen retrieval pretreatments for diagnostic immunohistochemistry Methods of antigen recovery vary in their usefulness in unmasking specific antigens in immunohistochemistry Comparison of the effects of microwave heating and high pressure cooking for antigen retrieval of human and rat Bc1-2 protein in formaldehyde-fixed, paraffin-embedded sections Superheating antigen retrieval

Drawbacks:   

Endogenous biotin staining as an artifact of antigen retrieval with automated immunostaining Retrieved endogenous biotin: a novel marker and a potential pitfall in diagnostic immunohistochemistry The enhanced reactivity of endogenous biotin-like molecules by antigen retrieval procedures and signal amplification with tyramine

 

Antigen retrieval: its significance and drawbacks in immunohistochemistry Antigen retrieval on formaldehyde-fixed paraffin sections: its potential drawbacks and optimization for double immunostaining

Electron Microscopy:       

An Antigen Retrieval Method Using an Alkaline Solution Allows Immunoelectron Microscopic Identification of Secretory Granules in Conventional Epoxy-embedded Tissue Sections An Evaluation of Antigen Retrieval Procedures for Immunoelectron Microscopic Classification of Amyloid Deposits Antigen retrieval of loricrin epitopes at desmosomal areas of cornified cell envelopes: an immunoelectron microscopic analysis A method for measurements of the efficiency of immunogold labelling of epoxy-embedded proteins subjected to different retrieval techniques Antigen Retrieval Trial for Post-embedding Immunoelectron Microscopy by Heating with Several Unmasking Solutions pH-dependent effect of heat-induced antigen retrieval of epoxy section for electron microscopy Increased level of immunogold labeling of epoxy sections by rising the temperature significantly beyond 100 degrees C in the antigen retrieval medium

Frozen Section Antigen Retrieval:    

Microwave superheating enhances immunocytochemistry in the freshly frozen rat brain Antigen Retrieval by Heating En Bloc for Pre-fixed Frozen Material A simple and sensitive antigen retrieval method for free-floating and slide-mounted tissue sections Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS)

Other Antigen Retrieval Methods:       

Immunoglobulin light chain staining in paraffin-embedded tissue using a heat mediated epitope retrieval method An optimal antigen retrieval method suitable for different antibodies on human brain tissue stored for several years in formaldehyde fixative One heat-induced antigen retrieval step allows the sequential detection of two antigens on the same slide of tissue fixed and enclosed in paraffin Brief, high-temperature heat denaturation (pressure cooking): a simple and effective method of antigen retrieval for routinely processed tissues A modified reduced-temperature antigen retrieval protocol effective for use with a polyclonal antibody to cyclooxygenase-2 (PG 27) A novel, simple, reliable, and sensitive method for multiple immunoenzyme staining: use of microwave oven heating to block antibody crossreactivity and retrieve antigens Microwave antigen retrieval for immunocytochemistry on formalin-fixed, paraffin-embedded post-mortem CNS tissue