was freshly prepared immediately before use; 10 ml of stock Solution B and 2 ml of glacial acetic acid ... Eer JM: Retepte fur die Fotografie. Halle (Salle) Verlag ...
Journal of Histochemistry & Cytochemistry http://jhc.sagepub.com/
A simple enhancement method for the silver-gold-intensified diaminobenzidine reaction in the light microscopic immunoperoxidase technique. S Goto, S Nagahiro, Y Ushio and W Hofer J Histochem Cytochem 1992 40: 1423 DOI: 10.1177/40.9.1506678 The online version of this article can be found at: http://jhc.sagepub.com/content/40/9/1423
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0022-1554/92/$3.30 Vol. 40,NO.9.pp. 1423-1425,1992 Printed in LXA.
The Joumal of Histochemistry and Cytochemistry Copyright 0 1992 by The Histochemical Society, Inc.
Technical Note
A Simple Enhancement Method for the Silver-Gold-intensified Diaminobenzidine Reaction in the Light Microscopic Immunoperoxidase Technique SATOSHI GOTO,’ SHINJI NAGAHIRO, YUKITAKA USHIO, and WALTER HOFER Department of Neurosurgery, Kumamoto University Medical School, Kumamoto 860, Japan (SG,SN,YU), and Electron Microscopy Laboratory, Max-Phnck-Institutefor Brain Research, Frankfurt-am-Main, Germany (WH). Received for publication December 23, 1991 and in revised form March 25, 1992; accepted April 13, 1992 (1T2542).
We describe a simple and sensitive method for enhancement of the silver-gold-intensified 3 3 -diaminobenzidine (DAB) reaction demonstrating peroxidase activity. After completing silver-gold intensificationof the preparationsimmunostained by the avidin-biotin-peroxidase method with DAB as the chromogen, the preparations were immersed in a so-
Introduction The silver-gold intensification of 3,3’-diaminobentidine(DAB) products is widely used in immunoperoxidasestaining (2,5-7). However, in some cases additional amplificationof the signal is required. A more sensitive method could enable determinations in immunocytochemistry with a lower concentration of primary antibodies. We report here a simple and sensitive procedure to enhance the silver-gold-intensified DAB reaction in an immunoperoxidase method.
Materials and Methods Tissue Preparation. An adult monkey (Papioanubis) was perfused with 4% formaldehyde in 0.1 M phosphate buffer, pH 7.4, and post-fixed in the same fixativefor 1 day. Thereafter, brain tissues were embedded in paraffin. Six-pm thick sections were prepared on a microtome and mounted on gelatin-coated glass slides. The sections were routinely deparaffinized and rehydrated and were processed for immunostaining. Immunostaining Procedure. Mouse monoclonal antibody to microtubule-associated protein 2 (MAP2; Sigma, St Louis, MO) was used as a primary antibody. To demonstrate the effect of uranyl enhancement on silver-gold-intensified materials, we intentionally reduced the concentration of primary antibody against MAP2 to 1:20,000, which was 100-foldlower than usual. The sections were incubated with antibodies to MAP2 in PBS containing 3% BSA for 24 hr at 4°C. To visualize bound antibodies, a con-
’
Correspondence to: Satoshi Goto, MD, PhD, Dept. of Neurosurgery, Kumamoto University Medical School, Kumamoto 860, Japan.
lution containing uranyl nitrate. This new method appeared to increase the sensitivity by at least one order of magnitude as compared with silver-gold intensification alone. (JHisrochem Cyrochem 40:1423-1425, 1992) KEY WORDS: Diaminobenzidine; Silver-gold
intensification;Immunocytochemistry; Uranyl nitrate enhancement.
ventional avidin-biotin-peroxidase complex (ABC) method (4) was carried out using the Elite ABC kit (Vector; Burlingame, CA) as described previously (3). DAB (Sigma) was used as a chromogen. Staining specificity was carefully assessed by examination of specimens with replacement by non-immune mouse IgG as a primary antibody. Silver-Gold Intensification Procedure. The silver-gold intensification for DAB reaction products was performed exactly as reported by Rossi et al. (6). Briefly, the immunostained sections were washed in sodium acetate (2% in distilled water) and then incubated in sodium thioglycolate (10% in distilled water) for 3 hr. After rinsing in sodium acetate they were incubated in a freshly prepared solution containing 2.5% sodium carbonate, 0.1% silver nitrate (Nakarai Chemicals; Kyoto, Japan), 0.1% ammonium nitrate (Nacalai Tesque; Kyoto, Japan), 0.5 % silicowolframic acid (Merck; Darmstadt, Germany), and 0.008% formaldehyde in distilled water, for 8 min at room temperature. Thereafter, they were quickly rinsed in acetic acid ( 2 % in distilled water), followed by rinsing in sodium acetate and incubation in gold chloride (Muto Pure Chemicals; Tokyo, Japan; 0.05% in distilled water). After rinsing in sodium acetate, the sections were immersed in sodium thiosulfate (Nacalai Tesque, 3% in distilled water) for 10 min and washed with distilled water. Uranyl Enhancement of Silver-Gold-intensified DAB Reaction. The silver-gold-intensified specimens were immersed in the developer, which was freshly prepared immediately before use; 10 ml of stock Solution B and 2 ml of glacial acetic acid were added to 10 ml of stock Solution A for 15 min at room temperature. When the desired grade of enhancement had been achieved macroscopicallyor light microscopically, the sections were quickly washed with 70% ethanol and dehydrated before mounting in entellan-xylene. Stock Solution A consisted of 1 g of potassium ferricyanide (Wako Pure Chemicals; Osaka,Japan) dissolved in 100 ml of distilled water. Stock Solution B consisted of l g of uranyl nitrate (Wako) dissolved in 100 ml of distilled water. 1423
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1424
GOTO, NAGAHIRO. USHIO, H O E R
I
A
Figure 1. Application of the uranyl enhancement for immunohistochemislry. On the same slide. Iwo sections from monkey brainstem were processed for MAP2 immunodainingand subjected to silver-gold inlensificationwilh or without uranyl enhancement (A,C) Silver-gold intensification only, (B,D) silver-gold intensification plus uranyl enhancemenl. Bars: A,B = 0 5 mm; C.D = 100 pm. Uranyl Enhancement of Silver-Gold-intensified DAB Reaction in a Model System. Two-pl aliquots from tenfold serial dilutions o f whole rabbit serum (Sigma) were blotted on a nitrocellulose membrane (Bio-Rad; Richmond. CA) and air-dried. The membrane was rehydrated and incubated in PBS containing 10% bovine serum albumin (Nakarai Chemicals) overnight at 4'C. lmmunosraining was then carried out by the ABC method with biorinylared anti-rabbit IgG (Vector) in the usual manner. The immunostained membrane containing the D A B reaction product was extensively washed with distilled water and subjected to silver-gold intensification with or without uranyl enhancement. as described above.
Results and Discussion We applied the uranyl amplification technique for the immuno-
A
@
Figure2. Uranyle n h a n c e " of sllver-golblntensified DAB m a i o n in a model system. (A) Silver+ld intensification plus uranyl enhancement; (B) silver-gold intensification only.
stained preparations taken from formalin-fixed. paraffin-embedded monkey brain. An additional treatment of uranyl enhancement could markedly increase the staining intensity macro- and microscopically as compared with silver-gold intensification alone (Fig ure 1). The final staining pattern obtained with the new technique was proportional to that of the silver-intensified DAB products, showing no apparent nonspecific background activity. As a result of the uranyl enhancement, the dark-brown deposits ofsilver-goldintensified DAB products tumed true brown, and several sites containing undiscernible amounts of them were stained light brown. Our preliminary experiments indicated that the final reaction was completed within 15 min of incubation and that an incubation time longer than 15 min caused high background activity, specific staining also being slightly increased. We haw applied this technique to sections from other species, such as rat and cat, and the same enhancement effect was obtained in those. In addition, from our experience this method is also applicable for routinely prepared. formalin-fixed, paraffin-embedded human materials. such as surgical specimens. Figure 2 shows the enhancement effect of the new method in a model system using nitrocellulose membrane. By the additional uranyl enhancement of silver-gold-intensified DAB reaction products, the detection limit appears to be raised at least tenfold in magnitude as compared to the simple silver method for DAB alone. The special method introduced here is an old photographic tcchnique used to develop contrast on negative film (1). As shown here, it can be successfully applied to silvcr-gold-intensified DAB precipi-
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1425
URANYL ENHANCEMENT TECHNIQUE
tates obtained by immunoperoxidase procedures. Uranyl enhancement should prove to be a simple and sensitive method for amplification of the DAB end-products in various immunostainings, such as immunocytochemistry.
Literature Cited 1. Eer JM: Retepte fur die Fotografie. Halle (Salle) Verlag, 1942
2. Gallyas F, Gorcs T, Merchenthaler I: High-grade intensification of the end-productofthe diaminobentidinereaction for peroxidasehistochemistry. J Histochem Cytochem 30283, 1982 3. Goto S, Hirano A, Matsumoto S: Subdivisional involvement of nigrostriatal loop in idiopathic Parkinson’s disease and striatonigral degeneration. Ann Neurol 26:766, 1989
in immunoperoxidasetechniques: a comparison between ABC and unlabeled antibody (PAP)procedures.J Histochem Cytochem 29577,1981 5. Liposits 2, Setalo G, Flerko B Application of the silver-gold intensified
3.3’-diaminobenzidine chromogen to the light and electron microscopic detection of the luteniting hormone-releasinghormone system of the rat brain. Neuroscience 13:513, 1984 6. Rossi F, van der Want JJL, Wiklund L, Strata P Reinnervation of cerebellar Purkinje cells by climbing fibres surviving a subtotal lesion of the inferior olive in the adult rat. 11. Synaptic organization on reinnervated Purkinje cells. J Comp Neurol 308536, 1991 7. van den Pol AN, Gorcs T Synaptic relationships between neurons con-
taining vasopressin, gastrin-releasingpeptide, vasoactive intestinal polypeptide, and glutamate decarboxylaseimmunoreactivity in the suprachiasmatic nucleus: dual ultrastructural immunocytochemistry with goldsubstituted silver peroxidase. J Comp Neurol 252:507, I986
4. Hsu S, &ne L, Fanger H Use of avidin-biotin-peroxidase complex (AEK)
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