Journal of Histochemistry & Cytochemistry

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A histological processing technique that preserves the integrity of calcified tissues (bone, enamel), yolky amphibian embryos, and growth factor antigens in skeletal tissue. W T Bourque, M Gross and B K Hall J Histochem Cytochem 1993 41: 1429 DOI: 10.1177/41.9.7689084 The online version of this article can be found at: http://jhc.sagepub.com/content/41/9/1429

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0022-1554/93/$3.30 The J o u d of Histochemistry and Cytochemistry Copyright 0 1993 by The Histochemical Society, Inc.

Vol. 41,No. 9,pp. 1429-1434,1993 Printed in US.A.

Technical Note

A Histological Processing Technique that Preserves the Integrity of Calcified Tissues (Bone, Enamel), Yolky Amphibian Embryos, and Growth Factor Antigens in Skeletal Tissue' WILLIAM T. BOURQUE,2 MICHAEL GROSS, and BRIAN K. HALL Department of Biology (WBBH) and Department of Surgery, Division of Orthopaedics (MG), Dalbousie University,Ha&&, Nova Scotia, Can&. Received for publication June 17, 1992 and in revised form October 5 , 1992; accepted November 16, 1992 (2T2716).

We have devised a proassing technique to embed calcified tissues, such as bone and tooth enamel, in paraffin, to preserve the delicate antigenic sites of molecules such as growth factors. The same technique, omitting the decalcification step, allows delicate tissues, such as axolotl embryos (Ambgstoma mexicantun)containinglarge yolk masses, to be easily handled during tissue promsing and to be serially sectioned. Specimens were all fued in periodate-lysine-paraformddehyde (PLP)fmtive at 5'C. Bone and teeth were decalcified in an EDTA-Gsolution at -4'C. Maintaining a temperature of Sac,the decalcified samples were then washed (with PBS, pH 7.2,under vacuum) to remove glycerol. Both the

Introduction The use of conventional fixatives such as neutral buffered formalin (NBF) and Bouin's, followed by routine paraffin embedding of tissues, destroys many delicate antigenic sites, whereas cryosectioning compromises morphological detail in the tissues (1).Many surface antigens are glycoproteins embedded in the lipid-containing plasma membrane and are especially susceptible to destabilization, followed by denaturation and/or mobilization, during tissue processing (2). With frozen-section immunohistdemistry, correlation of morphological and cytological features with immunophenotypeis difficult. Altematiwly, routine paraffii embedding destroysmany delicate antigens that might be detected by frozen sectioning. In theory, plastic embedding should be superior to either frozen or paraffin

Supported by an NSERC grant (BKH) and by the Orthopaedic Research Fund of the Victoria General Hospital (MG).

Correspondence tO: W.T. Bourque. Faculty of Dentistry. Dept. of P m t i v e Dental Science, Univ. ofManitoba, Winnipeg, Manitoba, Canada R3E OW2.

decalcified tissues and the yolky embryos were dehydrated through an ascending series of isopropanol and embedded in low melting-point paraffin under vacuum. Acidic fibroblast growth haor (aFGF)was located in cells of the expanded CambiallayerintheearlyftaautecalluseSofmalecD-1 mice, demonstrating retention of antigenic sites. The results reported here have not previously been obtained with existing processing and embedding techniques. (JHistdem Cytochem 41:1429-1434, 1993) KEY WORDS:Acidic fibroblast

growth factor; Immunoperoxidase;

Paraffinembedding;Fracture repair; Yolky embryos; Bone; Enamel.

sections for immunohistochemistry because of better antigenic and morphological preservation. In practice. however. ~lasticembedding is limited by technical difficulties. Water-soluble plastics such as glycol methacrylate apparently bind to amino-terminal portions of proteins during polymerization, masking antigenic epitopes. This masking can be reversed by trypsin or protease treatment of sections, but proteolysis @teTzr>estroys some antigens and compromises morphology (3). Bone is an inherently difficult tissue to work with histologically. Most histological techniques require some form of decalcification before bone can be sectioned (4). Bone itself acts as a reservoir of non-collagenous extracellular proteins, which include growth factor molecules such as acidic fibroblast growth factor (5), transforming growth factor$ ( 6 ) and insulin-like growth factors I and I1 (7). The identity of which types of cells and under which conditions these growth factors are produced in vivo is only beginning to be established. We report here modifications to several previously existing histological methods which, together, form a new technique for immunohistochemical study of calcified tissue and which provide excellent tissue integrity in serially sectioned decalcified tissues and yolky embryos. 1429


Materials and Methods Polyclonal rabbit anti-bovineacidic fibroblast growth factor (aFGF)was obtained from Upstate Biotechnology (Lake Placid, NY).Biotinylated antirabbit IgG was purchased from Vector Laboratories(Burlingame, CA). Natural bovine aFGF (containing bovine serum albumin as a carrier protein) was purchased from R & D Systems (Minneapolis, MN).

Harvest Of Tissues Healing Fractures. Male CD-1 mice (CharlesRiver; Montreal, Quebec, Canada), 4-6 months old, were subjected to a reproducible closed fracture procedure using a custom-designed fracture apparatus (8). The right tibia was fractured in seventeen mice. Five of these fracture-treated mice were used for histological processing and immunohistochemicallocalization of aFGF. The other 12 mice with fractured tibiae were used for the immunological detection of aFGF by Western blot analysis. At Day 5 post fracture, these animals were sacrificed and the fractured limbs removed. Fracture calluses at this stage of fracture repair consisted of the tibial shaft, bone marrow, proliferating cartilage-like cells of the cambial layer of the periosteum, and undifferentiated fibroblast-like mesenchymal cells. The 12 fracture-treated mice for Western blot analysis had only the fracture callus dissected and homogenized. The five fracture-treated mice for immunohistological study had their fractured tibiae removed and placed in freshly prepared PLP fixative (2% paraformaldehyde containing 0.075 M lysine and 0.01 M sodium periodate solution) as previously described (9). Murine Teeth for Histological Study. The mandibular left first molars were removed from six adult CD-1 mice and placed in freshly prepared PLP fixative. Axolotl Embryosfor Histological Study. Six Stage 36 axolotl embryos (purchased from the University of Ottawa axolotl colony) were placed in fresh PLP fixative. All of the fixed tissues were then placed in glass vials, placed in a rotating drum, and left for 24 hr at 5°C.

solutions: 0.01 M PBS containing 5% glycerol, 0.01 M PBS containing 10% glycerol, and 0.01M PBS containing 15% glycerol as previously reported (10). The tissues were then suspended by thread in 155 ml of EDTA-G solution at -4°C [14.50g EDTA (CloH14Nz0~NAZ.2H~O), 15 ml of glycerol, 85 ml distilled water. and solid sodium hydroxide (NaOH) added until a final pH of 7.3 was reached as previously described (lo)]. A hole was pierced in the plastic lid of a 200-ml glass jar and the thread attached to the tissue suspended from it. This E m - G solution was replaced every 7 days. Progression of decalcification was checked by piercing the tissue with a dissecting needle. Decalcification was complete after 16 days in the fractured tibia samples and 32 days for the mouse molars. The decalcdyingstep was omitted in the treatment of the axolotl embryos.

Washing of Decalcijtid Tissues Glycerol was removed from the calcified tissues by lengthy washing. Tissues were washed with a solution containing 15% sucrose and 15% glycerol in PBS at -4'C for 12 hr. After this, the washes alternated between 12 hr under vacuum at 5°C and 12 hr in a rotating drum at 5%. The washing solutions were as follows: 20% sucrose and 10% glycerol in PBS, 20% sucrose and 5 % glycerol in PBS, 20% sucrose in PBS, 10% sucrose in PBS, 5 % sucrose in PBS, and 100% PBS.

Dehydration and Parafin Embedding The fractured tibiae, mouse molars, and axolotl embryos were then dehydrated at 5 'C in a rotating drum through the followingseries of isopropanol solutions: two changes of 50:50 isopropanoUdHz0 for 1 hr each, one change of 60:40 isopropanoUdH20 for 1 hr, one change of 70:30 isopropanol/dH20 for 1 hr, one change of 80:20 isopropanolldH~0for 1 hr, one change of 90:lO isopropanol/dH*Ofor 1 hr, four changes of 100% isopropanolfor 1 hr each, one change of 5050 isopropanokhloroform for 1 hr, one change of 100% chloroform for 2 hr at - 2O'C. The tissues were then transferred to Paraplast X-sa low-melting-point paraffin, MP 56'C (Fisher; Montreal, Quebec, Canada). Four changes of wax (1 hr each) under vacuum were used before embedding in low-melting-point paraffin.

Processing of Tissues for Western Blot The 12 dissected calluses were placed in PBS, pH 7.2, containing 1:lOOO PMSF (Sigma; St. Louis,MO). Th9 were then homogenized in a Kinematica tissue homogenizer (Brinkman Instruments; Westbury, NY)for 45 sec. The homogenate was then spun for 20 min at 3000 rpm in a Beckman GP centrifuge (Beckman Instruments; Palo Alto, CA) to remove solid material. A separation column was constructed consisting of a Pharmacia column (40cm long x 2 cm diameter) packed with Sephadex G-200 (Pharmacia; Uppsala, Sweden). The column was standardized by passing proteins of known molecular weights down the column and collecting the eluent. The collected eluent was analyzed spectrophotometricallyto determine which fractionscontained the standard molecular weight proteins. The tissue homogenate was loaded into the column and eluted with Tris buffcr, pH 8.0, containing PMSF. Only the eluted fractions containing proteins of less than 43 KD were collected. This eluent was further concentratedwith a Centricon10centrifuge concentrator (Amicon; Danvers. MA) which allowed proteins of less than 10 KD to be removed from the solution. The retentate solution (containing the 43-10 KD protein fractions)was further concentrated with a SpeedVac SVC 100 (Savant; Farmingdale, NY).The final volume of the retentate was 1 ml.

Decalct3cation of Tissues After fmtion, the five fractured tibiae and the adult molar teeth were washed for 12-hr intervals at 5°C (in a rotating drum) in the following series of

Trimming of Blochs The tissue blocks were trimmed down to the tissue surface. The block (tissue surface down) was placed in a dish of distilled water for 20 min, the tissue surface blot-dried, and the block was placed in a freezer at - 20% for 20 min. The block was then placed in a microtome and cut at a thickness of 5 pm.Tissue sectionswere floated in a water bath at 48'C and placed on poly-L-lysine-coated glass slides. Sections of fracture callus, teeth, and axolotl embryos were stained with the HBQ method. This method gives excellent distinction between bone, cartilage, and connective tissue (11).

Immunohistochemical Method The paraffin sections were deparaffinized and stained by the avidin-biotin-peroxidase complex (ABC)hmunopcroxidase technique according to Hsu et al. (12), with some modifications. Endogenous peroxidase activity was blocked by immersing the sectionsin 3% hydrogen peroxide for 5 min. followed by washing in running water for 5 min and then flooding with PBS for 5 min. The sections were then incubated with 100% (vlv) normal goat serum for 30 min at room temperature (RT) to block nonspecific binding. The sections were drained well and then incubated overnight at 4'C with primary antibody diluted with PBS (containing 0.1% bovine serum albumin) in the ratio of 1:SOO. After washing with PBS, the sections were incubated with secondary antibody (biotinylated goat anti-rabbit IgG)


1431

TECHNIQUE OF GROWTH FACTOR DEMONSTRATION IN BONE

:

diluted with PBS in the ratio of 1:200 for 30 min at RT. The sections were then washed with PBS and incubated with avidin-biotin-peroxidase complex ABC Elite Kit PK-6100 (Vector) for 30 min at room temperature. After washing with PBS. brown pigmentation was produced by 2-min treatment with 3,3'-diaminobenzidine (DAB) (Sigma; St Louis, MO) (1 DAB tablet dissolved in 20 mlO.05 M Tris-HCI buffer, pH 7.6, containing 0.005% hydrogen peroxide). As a positive control, natural bovine aFGF was added to an agarose gel along with a tissue homogenate of Day 5 post-fracture calluses and run as a Western blot. As a negative control, PBS was substituted for the primary antibody in the immunohistological method.

?I

..

Western BZot

1

T

This procedure was standard. The aFGF (15 PI) and 45 PI fracture callus retentate were electrophoresed in a 12.5% SDS-PAGE gel. The protein was transferred to nitrocellulose paper with pore size 0.45 wm (Schleicher & Schuell; Keene, NH) in transfer buffer ( 2 5 mM Tris, 192 mM glycine, p H 8.3, with 20% methanol). Overnight transfer was performed in a Bio-Rad (Melville, NY)TransBlot transfer apparatus (30 mA constant current). Nonspecific binding was blocked by washing the nitrocellulose in 5 % gelatin solution for 30 min. Nitrocellulose was washed in Tween-Tris buffered saline (TTBS) (10 mM Tris-HCI, p H 7.4. 140 mM NaCI, 0.1% (v/v) Tween 20). then incubated with anti-aFGF antibody for 1.5 hr at RT. This was followed by washing in TTBS and incubation in biotinylated goat anti-rabbit secondary antibody for 30 min. After rinsing with TTBS, the nitrocellulose was incubated in avidin-biotin complex conjugated to alkaline phosphatase (ABC-AP kit AK-5000; Vector). The nitrocellulose was then washed with TTBS containing a high salt concentration to remove nonspecific binding. A final rinse in TBS was followed by the p,aduction of pigmentation using BCIP and NBT (Sigma). The reaction was stc;oed by rinsing the nitrocellulose in distilled water.

Summary of Preservation Technique

* *

FM c

T

Calcified Tissue. Samples were placed in fresh PLP fixative and fixed at 5'C for 24 hr, then washed in PBS containing increasing concentrations of glycerol (5%. lo%, 15%). The samples were decalcified in EDTA-G solution (pH 7.3) at -4°C. After decalcification, samples were washed (alternating between vacuum extraction and washing at atmospheric pressure) at 5'C initially in PBS-glycerol, then PBS sucrose, and finally PBS, dchydrated through an ascending series of isopropyl alcohols at 5'C. and cleared in chloroform at - 2O'C. Samples were vacuum-infiltrated and embedded in low-melting-point paraffin at 56'C. On cooling, the paraffin block was trimmed and the tissue surface was immersed in a tray of distilled water at RT for 10 min. The tissue surface was then blotted and the block was placed at -20°C for 20 min. Soaking and freezing were repeated as necessary to continue serial sectioning. Paraffin sections were floated on a water bath and mounted on poly-L-lysine-coated slides. Non-calcified Tissues. Samples were fiied in fresh PLP fixative at 5'C for 24 hr and washed in PBS (pH 7.2). Samples were then dehydrated and embedded. Sections were cut and mounted as described.

-

CL IC

Figure 1. (a) Fractured tibia 5 days post fracture stained by the HBQ method. The shaft of the tibia (T) is visible, as is the fracture (F). (b) A higher-power photomicrograph of the area outlined in a. The cells of the cambial layer (CL) of the periosteum and fibroblast-like mesenchymal cells (FM) are clearly visible. (c) Anti-aFGF antibodies were applied to a serially adjacent tissue section to theone stained with HBQ in a. Photomicrographedunder high power, positive anti-aFGFstaining (arrowheads) in cells of the cambial layer wasdemonstrated. Original magnifications: a x 30; b,c x 410. Bars: a = 330 pm; b,c = 25 pm.


1432

BOURQUE. GROSS, HALL

.. -1

'2;

*'q

1.

Figure 2. (a) Photomicrographof a portion of a mouse mandible containing the lower left molar. The section displays teeth 0surrounded by periodontal ligament (PL) and alveolar bone (AB) stained with the HBQ method. (b) A higher-power photomicrograph of the area outlined in a. The section demonstrated excellent tissue integrityof the contacts between the periodontal ligament, teeth, and alveolar bone. Original magnifications: a x 30; b x 260. Bars: a = 330 pm; b = 40 pm. Figure 3. (a) Photomicrograph of axolotl embryo. The section was stained by the HBQ method. Note the absence of tearing in the yolk sac (YS).Also excellently preserved are the heart (H), brain (B), and spinal cord (SC). (b) A higher-power photomicr9graph of the area outlined in a. The section demonstrated excellent preservation of detail of fine structures like the notochordal membrane (NM). Original magnifications: a x 30; b x 410. Bars: a = 330 pm; b = 25 pm.

Results Tissue Integrity The Day 5 post-fracture callus tissue sections showed no evidence of tearing. The decalcified bone and loose fibroblast-like mesenchymal tissue remained intact. In addition, there was no evidence of distortion due to shrinkage between the tibia, cambial layer of the periosteum, and fibroblast-like mesenchymal cells (Figures la and Ib). The tissue sections of the lower mandible containing the left first molar also showed no evidence of tissue tearing. The spatial relationships of enamel to periodontal ligament and periodontal ligament to alveolar bone were well preserved. High-power photomicrographs of these tissue sections also revealed little distortion due to cell shrinkage as a result of decalcification and tissue processing (Figures 2a and 2b). The transverse sections of the Stage 36 axolotl embryos again showed no evidence of tearing. The entire embryo, including the

yolk sac, remained intact. The cells of the embryonic tissues maintained their spatial relationships to one another. Even very delicate tissue structures, such as the notochordal membrane, were well preserved in these tissue sections (Figures 3a and 3b).

Immunohistochemical Studies The anti-bovine aFGF antibody recognized the pure bovine aFGF in Lane 2 of the Western blot (Figure 4) and also recognized a protein in the fracture callus retentate in Lane 1. The fracture callus retentate band occurred close to the positively stained bovine aFGF band; difference in the molecular weights may be due to species variation. When this antibody was applied to tissue sections for immunohistochemical analysis, cells in the expanded cambial layer of the periosteum adjacent to the fracture site stained intensely. The staining was very specific and was confined to these cambial layer cells in the Day 5 post-fracture callus (Figure IC).The negative control contained no nonspecific staining.


TECHNIQUE OF GROWTH FACTOR DEMONSTRATION IN BONE

1

1433

ture (15). Mori et al. (10) decalcified small bone samples at pH 7.1-7.4 and at a temperature of -5°C. The calcium was removed from the bone in these samples with minimal damage to surface antigens. In the present study, problems arose when we attempted to section Day 5 post-fracture calluses according to the method of Mori et al. (10). The glycerol used to depress the freezing point of the decalclfying solution below 0°Cwas not readily removed from muscle, dense bone, or enamel. This problem was overcome by alternating washing in PBS at 5'C with vacuum extraction of the glycerol into PBS at 5°C. A gradation of the washing solutionsfrom PBS-glycerol to PBS-sucrose to 100% PBS prevented cell rupture due to osmotic shock.

2

Effects of Paraffin Embedding

4 Figure 4. A Western blot probed with anti-bovine aFGF. Lane 1 contains retentate from Day 5 post-fracturecalluses. Lane 2 contains pure bovine aFGF and bovine serum albumin as a carrier protein. Bars mark positions of 18 and 67 KD molecular weight markers.

Discussion Effects of Fimtion Cell surface antigens are very delicate proteins which can be easily denatured during routine fixation and decalcification. The PLP fixative devised by McLean and Nakane (9) links carbohydrate moieties without excessive binding to protein antigenic sites. Pollard et al. (13) demonstrated optimal cell surface antigenic preservation on T-cells embedded in paraffin. They used a modified PLP fmtive and processed the tissue samples at 4°C through isopropanol, chloroform, to low melting-point paraffin. However, Pollard et al. (13) did not work with bone, enamel, or yolky tissue. Mori et al. (10)demonstrated excellent retention of antigenicity for cell surface antigens on lymphocytes and macrophages when cryosectioning PLP-fixed samples of destructive lesions from bone tissues. Because of these encouraging results, the fracture calluses, lower mandibular molars, and axolotl embryos were all fixed in PLP fixative and processed at 4°C using isopropanol as the dehydrating agent.

Effects of Decalcz-cation Cells in decalcified bone typically become shrunken, and many details of the bone matrix become blurred because of swelling of the osteocollagenousfibers by the decalcifying agents and processing procedures used to decalcify bone (14). It has been reported that PLP-fixed tissues give excellent preservation of tissue architec-

The method of Mori et al. (10) for cryosectioning the decalcified tissues still presented problems even after extensive washing of the decalcified bone and enamel with PBS. The cryostat used to section these tissues produced torn sections of irregular thickness. In addition, the sections routinely lifted off poly-L-lysine coated glass slides. Pollard et al. (13)conducted an extensive study of the dfects that dehydrating agents and processing temperatures have on the preservation of surface antigenic sites on T-cells. These authors found that the use of isopropanol as a dehydrating agent, processing of tissue at low temperatures, and use of low melting-point paraffin vastly improved the preservation of surface antigenic sites over dehydration in ethanol, processing of tissues at room temperature, and embedding in higher-temperature paraffin. It was decided to infiltrate the decalcified bone and enamel, as well as the axolotl embryos, with low melting-point paraffin. The tissue specimens were all dehydrated through an ascending series of isopropanol solutions at 5°C. cleared in chloroform at -20'C. and vacuum-infiltrated with low melting-point paraffin. The use of a vacuum was found to be essential for the removal of glycerol from decalcified specimens and for the proper infiltration of the paraffin into bone, enamel, and yolk sac. An additional step involved trimming the block of embedded tissue to expose the tissue surface and then placing this exposed surface in a petri dish containing distilled water for a period of 20 min. After this, the block was placed face down in another petri dish and then inserted into a freezer for 20 min. This additional step dramatically improved the ability of the embedded tissue to be serially sectioned. Omitting this step resulted in torn tissue sections. This procedure differs from the routine lowering of the tissue block surface temperature with ice before cutting. The bone, molars, and embryos used in this method had to be rehydrated by soaking in distilled water before cutting, otherwise they tore during sectioning. The rehydrationstep had to be followed by freezing the rehydrated tissue. Omitting the freezing step resulted in the tissues becoming soggy and unable to be cut.

Capacity of the Processing Procedure for the Immunobcalization of aFGF The present procedure for fixing, decalcifying, processing, and embedding calcified tissues clearly demonstrates the presence of aFGF in Day 5 post-fracture calluses. The immunohistochemical staining was both specific to the cells of the cambial layer and dis-


1434

BOURQUE, GROSS, Hw

tinct. Studies by Gospodarowicz et al. (16) and Wellmitz et al. (17) have shown that administration of aFGF to amputation stumps of adult frog limbs and to knee-joint injuries in rabbits induces the proliferation of undifferentiated cells and chondrocytes.Jingushi et al. (18) also administered aFGF into rat fractures and reported a marked increase in the size of the cartilaginous soft callus. These studies establishedaFGF as a marker for cell proliferation. Hauschka et al. ( 5 ) have reported that aFGF can be isolated from bone matrix. It therefore appears that aFGF is also a constitutive part of bone tissue. However, since only the cells of the expanded cambial layer stained positive for aFGF, it is possible that in response to injury bone can produce significantlyincreased amounts of aFGF, possibly to induce the proliferation of cells involved in the early stages of fracture repair (i.e., fibroblast-likemesenchymalcells and chondrocytes). The ability of the present technique to preserve other growth factor antigens has also been tested. To date IGFI,EF-P, and PDGF have all been successfully detected in the fracture site during the stages of fracture repair. These other growth factor findings are described in a manuscript that is in the processes of being completed. In sections containing teeth, the periodontal ligament remained attached to both enamel and alveolar bone. Individual cells could be readily recognized in these tissues, which may facilitate the localization of antibodies. The preservation of tissue integrity and therefore preservation of tissue relationships (e.g., tooth to periodontal ligament, alveolar bone to periodontal ligament) (Figure 2b) is such that morphometric analysis could readily be performed on such tissues. The preservation of antigenicity demonstrated by the preservation of aFGF antigens in fracture calluses suggests enhanced preservation of antigens in dental tissue, which may be a valuable asset for dental research. For example, the activity of growth factor molecules and also matrix molecules could be investigated in greater detail owing to the excellent preservation of tissue architecture and antigenicity. The preservation of tissue detail in embryonic tissues, such as amphibian embryos, suggested improved preservation of antigenic sites on these tissues. Previous tissue processing procedures for yolky embryos were very labor intensive and involved the use of harsh chemicals (19). These harsh chemicals would have serious detrimental effects on the delicate antigenic sites on molecules such as growth factors. With the preservation ofsuch fine structuresas the notochordal membrane, immunohistochemicalstudies using this new processing procedure may lead to new insights into the activities of growth factors and other delicate molecules in embryonic development. In addition, this technique would be ideal for studying tissue or organ development.

Literature Cited 1. Stein H, Heryet A, Gatter KC, Mason DY. Freeze-dried paraffin-

embedded human tissue for antigen labeling with monoclonal antibodies. Lancet 1984;ii:71 2. Holgate CS, Jackson P, Pollard K. h n n y D, Bird CC. Effect of fixation

on T and B lymphocyte surface membrane antigen demonstration in paraffin processed tissue. J Pathol 1986;149:293 3. Casey lT,CousarJB, Collins RD. A simplified plastic embedding and immunohistologic technique for immunophenotypic analysis of human hematopoietic and lymphoid tissues. Am J Pathol 1988;131:183

4. Humason GL. Animal tissue techniques. 4th ed. San Francisco: WH Freeman, 1979 Hauschka PV. Mavrakos AE, Iafrati MD, Doleman SE, Klagsbrun M. 5. Growth factors in bone matrix: isolation of multiple types by affinity chromatography on heparin-sepharose. J Biol Chem 1986;261:12665 6. Seyedin SM, Thomas TC,Thompson AY, Rosen DM, Piez KA. Purification and characterizationof two cartilage-inducingfactors from bovine demineralized bone. Proc Natl Acad Sci USA 1985;82:2267 7. Frolik CA, Ellis LF, Williams DC. Isolation and characterization of insulin-like growth factor-I1from human bone. Biochem Biophys Res Commun 1988;151:1011 8. Bourque WT. Gross M, Hall BK. A reproduciblemethod for the production and quantification of the stages of fracture repair. Lab Anim Sci 1992;42:1

Nakane PK. Periodate-lysine-paraformaldehyde fixative: 9. McLean JW, a new fixativefor immunoelectron microscopy.J Histochem Cytochem 1974;22:1077 10. Mori S,SawaiT, Teshima T, Kyogoku M. A new decalclfyingtechnique for immunohistochemicalstudies of calcified tissue, especially applicable to cell surface marker demonstration.J Histochem Cytochem 1988;36111 11. Hall BK. The role of movement and tissue interactionsin the develop-

ment and growth of bone and secondary cartilage in the clavicle of the embryonic chick. J. Embryo1 Exp Morpho1 1986;93:133 12. Hsu S, Raine L, Fanger H. Use of the avidin-biotin-peroxidase com-

plex (ABC) in immunoperoxidasetechniques: a comparison between ABC and unlabeled antibody (PAP)procedures. J Histochem Cytochem 1981;29:577

13. Pollard K, LUMYD, Holgate CS,Jackson P, Bird CC. Fixation, processing, and immunochemicalreagent effects on preservation of T-lymphocyte surface membrane antigens in paraffin-embedded tissue. J Histochem Cytochem 1987;35:1329

14. Leeson CR, Leeson TS, Paparo AA. Text book of histology. 5th ed. Toronto: WB Saunders, 1985 15. Collins LA, Poulter LW, Janossy G. The demonstration of cell surface

antigens on T cells, B cells and accessory cells in paraffin-embedded human tissue. J Immunol Methods 1984;75:227 16. Gospodarowicz D, Rudland P, Lindstrom J, Benirschke K. Fibroblast

growth factor: its localization,purification, mode of action, and physiological significance. Adv Metab Disord 1975;8:301 17. Wellmitz B, Petzold E, Jentzsch KD, Heder G, Buntrock P. The effect of brain fraction with fibroblast growth factor activity on regeneration and differentiation of articular cartilage. Exp Pathol 1980;18:282

Acknowledgments

18. Jingushi S, Heydemann A, Kana SK, Macey LR, Bolander ME. Acidic

We wish to thank Dr Bill Pohajdak, Dr Tom Mclbe, and Brian Dixon for helpfil suggestions in running the Western blot and Sephadex column. We wouldalso like to thank the stajjfof the Specialstains Section, Department of Pathology, Victoria General Hospital for their helpfulsuggestions in performing the immunoperoxidase staining.

fibroblast growth factor (aFGF) injection stimulates cartilage enlargement and inhibits cartilage gene expression in rat fracture healing. J Orthop Res 1990;8:364 19. Pantin CFA. Notes on microscopical techniques for zoologists. New York: Cambridge University Press, 1960
