MICROSCOPY RESEARCH AND TECHNIQUE 20:274-280 (1992)

Medium Temperature Epoxy Resin for Immunocytochemistry: Quetol 651 With Water ANDRfi R.ABAD Department of Plant Pathology, University of Minnesota, St. Paul, Minnesota 55108

KEY WORDS

Epoxy resin, Acrylate and methacrylate resins, Protein A-Gold, Stereology, Labeling intensity

ABSTRACT The addition of 1%water to the epoxy resin Quetol increased the labeling intensity of the sample. The significant decrease of the curing temperature of the epoxy resin may assist in preservation of antigens. Water may also reduce the cross-linkage of the resin allowing more antigen to be available to the antibodies. The modified Quetol resin is an option for use in immunocytochemistry studies. INTRODUCTION Absorption of colloidal gold by protein A (Romano and Romano, 1977) offers the possibility to attach the protein A-gold complex to the Fc region of antibodies. The electron dense gold particle of the protein A-complex allows in situ localization of antigen-antibody reactions and can be observed with electron microscopy. This technique has permitted the study of cellular proteins. Immunocytochemistry, using this system, has gained wide attention and use. The success of the technique, assuming a well-characterized antibody, is dependent upon the survival of the antigen throughout tissue processing. This includes the variables of fixation, embedding media, and curing temperature. All of these variables must be considered to maximize antigenicity. Preservation of sample antigenicity does not imply retention of adequate ultrastructure (Bendayan et al., 1987).The compromise between these two objectives of sample preparation must be assessed in every study involving antigen labeling. The primary fixative, either glutaraldehyde or paraformaldehyde, alone or in combination, should be investigated to determine effects on retention of sample antigenicity (Paiement and Roy, 1988). Omission of osmium tetroxide as a fixative and stain is preferable since the metal masks the antigen. However, the omission of osmium fixation can create difficulties. Extraction and denaturation during dehydration and embedding of the non-osmicated sample may occur, introducing artifacts into the specimen (Weibull et al., 1983). The ultrastructure of the sample prepared without osmium may not be adequately preserved and osmium tetroxide must be used. Restoration of some antigenicity of osmium tetroxide treated samples can be accomplished with sodium metaperiodate on grid prior to immunolabeling (Bendayan and Zollinger, 19831. Several non-epoxy resins have been introduced for immunocytochemal study. These fall into two categories: the methacrylate and the acrylic resins. Several low temperature embedding methacrylate resins, glycol methacrylate (Leduc and Bernhard, 1967), low acid glycol methacrylate (Cole, 1984), Lowicryl K4M (Car-

0 1992 WILEY-LISS, INC.

lemalm et al., 19821, and the acrylic resin LR white (Newman et al., 19821, have been developed. There are drawbacks associated with the use of the methacrylate and acrylic resins. Microtomy of samples embedded in these resins is difficult. Consistency of resin blocks is frequently too brittle and the block face has a tendency to absorb water. Sections often contain small perforations which render the sections mechanically unstable to the electron beam. This causes severe drifting andlor collapse of sections. The poor resolution typically associated with the use of non-epoxy resins can represent a major source of compromise between good retention of antigenicity and adequate resolution. However, methacrylate or acrylic resins are required for the preservation of some antigens (Bendayan and Shore, 1982).In these cases, the antigens often are heat labile and do not survive the high curing temperature, typically 60°C or above, necessary for the polymerization of epoxy resins. In addition, it has been suggested that a destructive interaction may occur between sample proteins and the chemical components of the epoxy resin which also inhibits the preservation of antigenicity during infiltration and embedding of the sample (Stephens et al., 1982). Epoxy resins such as Epon 812 and Quetol651 have been used successfully in immunocytochemistry (Benhamou et al., 1985; Blanchette et al., 1989; Roth et al., 1981). In an immunocytochemical study comparing the epoxy resin Epon 812 with the non-epoxy resin Lowicryl K4M, Roth et al. (1981) observed no increase in the specific labeling intensity of their antigen between the two media. The background labeling, however, was reduced with the methacrylate resin Lowicryl K4M. Bendayan et al. (1987) have shown that the labeling intensity of amylase in pancreatic zymogen granules in samples embedded in Epon, Lowicryl K4M, or LR White was similar.

Received June 13, 1990 accepted in revised form June 13, 1991. Address reprint requests to Andre R. Abad, Department of Plant Pathology, University of Minnesota, 495 Borlang Hall, St. Paul, MN 55108.

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Addition of water to Lowicryl K4M (Bendayan et al., 1987) and to LR White (Newman and Hobot, 1987) improves the intensity of the labeling. An increase in labeling indicates a better preservation of the antigen. The survival of the antigen may be the result of protection by water from the denaturation effect of solvents. Alternatively, the background labeling may be reduced. The relief surface of the section may also be increased, exposing additional antigens to the antibodies (Kellenberger et al., 1987). The following investigation was undertaken to modify the embedding protocol of Quetol 651 (Abad et al., 1988; Kushida, 1974) to decrease the polymerization temperature of the resin and to increase the preservation of sample antigenicity by the addition of water to epoxy resin. MATERIALS AND METHODS Resin Preparation Water was added in increments of 0.5%by volume to Quetol (from 0.5 to 4%). The Quetol formulation used was Quetol651: 15 g, NMA: 20 g, NSA: 10 g, DMP-30: 0.6 g. With the addition of water, the resin initially acted like an emulsion (as with water and oil); however, with further mixing incorporation into the resin was achieved. The modified resin (Quetol plus water) was cured with temperatures ranging from 40°C to 74°C to obtain blank blocks. Sample Preparation The fungus Phanerochaete chrysosporium BKMF-1767 was grown on malt yeast extract media for 5 days a t room temperature. Mycelial masses were carefully removed and fixed with 1.5% glutaraldehyde in sodium cacodylate buffer at pH 7, 0.1 M for 2 hr. The hyphae were rinsed several times with buffer and divided into three treatments. Control 1treatment was processed via the standard protocol with 1%osmium fixation, water rinse, acetone dehydration up to loo%, Quetol embedding, and polymerization at 74°C. Control 2 treatment was treated the same as control 1 except for the omission of the osmication step (Table 1). The experimental treatment was dehydrated with acetone up to 95% (95 ml of 100% dry acetone + 5 ml of distilled water) infiltrated with 1/1ratio acetone 95%Quetol (Quetol without water) and embedded with 1% water added t o Quetol (Table 2). Sections 90 to 100 nm were collected with 200 mesh nickel grids and processed for immunocytochemistry.

TABLE 1. Standard Quetol embedding protocol, controls 1 and 2 Minimum time required

SteD 1.Fix sample in 1.5%glutaraldehyde cacodylate buffer at pH 7.2, 0.1 M 2. Rinse in buffer (2 x ) 3. Osmium (1%)in buffer' 4. Rinse in water 2 x ',' 5. Dehydrate with acetone 50% 75%

100% 100(dry) 100(dry) 6. Infiltration in quetol 50% quetoliacetone (1:l) 75% quetollacetone (3:l) 100%quetol 100%quetol 7. Quick change of resin; polymerization at 74°C

90 min

10 min 60 min 10 min 10 min 10 min 10 min 20 min 20 min 60 min 60 rnin 90 min 90 min 8 hr

'Step 3 and 4 were omitted for control 2. *For post-staining: 5 1 uranyl acetate in water from 2 to 8 hr, followed by three rinses with distilled water of 15 min each, then step 5.

TABLE 2. Quetol 1% water embedding protocol II, experimental treatment Minimum time required

Step 1. Fix sample in 1.5% glutaraldehyde cacodylate buffer at pH 7.2, 0.1 M 2. Rinse in buffer (2 x ) 3. Osmium (1%)in buffer' 4. Rinse in water 2 x 5. Dehydrate with acetone 50% 75% 95% 95% 6. Infiltration in quetol 95% acetone/quetol (im3 1%water in 100%quetol 1%water in 100%quetol 7. Quick change of resin; polymerization 74°C 58°C 48°C 40°C 3''

90 min

10 min 60 rnin 10 min 10 min 10 min 20 min 20 min 60 rnin 60 min 90 min 8 hr 8-12 hr 12-36 hr 24-48 hr

'Step 3 and 4 were omitted for group 4. 'Post-staining if desired after osmium: Uranyl acetate 5% in water for 2 to 8 hr at room temperature,followed by three washes of 15 min each, then proceeded by step 5. 3Quetol resin without water. Acetone prepared with 100%dry acetone.

The sections were floated on a drop of 0.1 M PBS buffer, pH 7.2, containing 1%NaN3 and 1%cold water Immunocytochemistry fish gelatin (Sigma G-7765) for 10 min. Sections were Polyclonal antibodies to three fungal secreted en- then transferred to a drop of buffer with an empirically zymes were used. Polyclonal antibodies raised in New determined concentration of primary antibody directed Zealand rabbit against xylanase enzyme, endo-l,4-p- toward the enzyme for 90 min at room temperature. glucanase I1 (EG 11) and 1,4-p-D-glucan cellobiohydro- The sections were rinsed thoroughly and floated on a lase enzymes, purified from Aureobasidium pullulans drop of protein A-gold complex in buffered solution Y-2311 (Leathers, 1986) and Trichoderma reesei (PBS pH 7.2,O.l MI for 30 min. Colloidal gold solutions (QM94141, respectively, were used to locate the site of were prepared by the Frens' method (1973) and conjuthe enzyme within the fungal cell. Ligninase antibody gated to protein A (Bendayan, 1984a; Roth et a]., 1978). to H8 from Phanerochaete chrysosporium BKM-F-1767 Grids were washed with 2 x distilled water and al(a generous gift by R.T.Farrell, Repligen-Sandoz Re- lowed to dry. Controls were as follows: l)the antibody search Corp, Lexington, MA 02173) was also used. All step was omitted, 2) the antibodies were absorbed by antibodies were tested for specificity with ELISA. addition of the enzyme for 48 hr at 4°C.

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The ability of the modified resin to increase the preservation of sample antigenicity was determined with antibodies raised against the several enzymes. The final dilution of the antibodies was maintained throughout the experiment a t 1:lOO. All sections of the three treatments were processed simultaneously with the same solutions to ensure comparable results.

Stereology Diameter changes created by possible shrinkage/ swelling of hyphae during processing among the sample preparations was studied in the three treatments. Micrographs were taken randomly at 8,000 x and the diameters of 20 hyphae were measured for each treatment. After the immunocytochemistry labeling, all of the hyphae of a randomly selected area of a section (one square per section per 200 Ni grid) were micrographed at 12,000-15,000 x . One grid of two different blocks per treatment provided at least the minimum of 15 hyphae chosen to evaluate the labeling intensity. The negatives were printed with an increase of magnification of 1.3 x . The surface area was calculated with the point counting technique (Gundersen and IZlsterby, 1980; Gundersen et al., 1988). The grids used had points separated by 13 mm. The formula employed was as follows: surface area (pm') = (13,000 p d t h e magnification)' x number of points encompassed by the sample. The same formula was used to calculate the background area (area outside the hyphea) and the area encompassed by the hyphae. Gold particles were counted in both hyphal and background areas. The number of gold particles per pm' was obtained by dividing the number of counted gold particles by the calculated area. The background representing unspecific labeling, if any, was subtracted from the sample labeling.

ter to the resin softens the final consistency of the blocks. During sectioning, sections expanded in size as they floated on the surface of the water in the diamond knife boat. When viewed with the electron microscope, sections taken from blank blocks of the modified resin were stable under the electron beam. The addition of 2.0% water to the resin seems to be the upper limit in which usable blocks can be produced. Modified Quetol will polymerize a t 45"C, but the time (48 to 72 hr) required for polymerization was excessive. At 48"C, the blanks blocks were cured within 12 t o 36 hr. For this study, the concentration of 1%water in Quetol and a curing temperature of 48°C was chosen to evluate the modified resin for use in immunocytochemistry. Hyphae from the different treatments were evaluated for the preservation of ultrastructure. The control 1 treatment (with osmium and cured at 74°C) represents the best possible resolution of the sample without post-staining (Fig. la). Both the membranes and ribosomes were well identified. Control 2 and the experimental samples without osmium (cured a t 74°C and 48"C, respectively) lacked preservation of ultrastructural resolution of the membranes (Fig. lb). The polyclonal antibodies were evaluated for their respective specificity with a liquid ELISA test. Xylanase antibodies were specific only to the xylanase enzyme and no cross-reactivity was observed with the other enzymes of the study. The ligninase polyclonal raised against the H8 lignin peroxidase enzyme shows a range of reactivity toward all lignin peroxidase and manganese-dependent peroxidase enzymes. Fungal enzymes of these classes have many similar polypeptides and the polyclonal reflects the common epitopes. In this study, ligninases should be considered to represent all classes of Mn-dependent peroxidases and ligninases peroxidase enzymes. Cross-reactivity between endoand exocellulases antibodies was minimal. To observe any possible swelling and/or shrinkage of hyphae prepared by the three treatments, the average hyphal diameter was calculated. Twenty specimens were recorded for each sample preparation. Within the Quetol system, no significant change for this particular morphological marker was found. The diameters in micrometers for 74°C/osmium, 74"C/no osmium, 48"C/no osmium were 2.40 f 0.15, 2.57 0.12, 2.47 2 0.16, respectively. After labeling the hyphae of each treatment using the polyclonal antibodies described, a stereology study was conducted (Table 3). An example of the difference in labeling intensity between control 1 (osmium and 78°C) and the experimental treatment (no osmium and 48°C) is given (Fig. 2a,b) using the lignin peroxidases antibody.

RESULTS Two characteristics of the epoxy resin were examined: the ability of Quetol to absorb water and the possibility of decreasing the curing temperature. The water-absorbing ability of Quetol was studied by adding varying amounts of water to aliquots of the resin and observing its polymerization into blank blocks a t 74°C. The final concentration of the water in the resin aliquots ranged from 0.5% to 4.0%. The resin was unable to cure to solid blocks if the concentration of water added was greater than 2.5%. However, acceptable blank blocks were produced with 2.0%water added to the resin. The hardness of the cured modified resin was softer with the concentration of 2.0%water compared with less than 1%or no addition of water. Aliquots of the modified resin with water concentraDISCUSSION tions of 0.5%, 1.0%, 1.5%, and 2.0% were polymerized with decreasing curing temperatures to establish the Typically, epoxy resins do not polymerize if residual lowest possible polymerization temperature. At 45"C, water is present. However, this investigation indicates the modified resin could be cured to solid blanks; how- that an epoxy resin, Quetol651, can absorb water. The ever temperatures below 45°C did not result in poly- benefits of the addition of water are twofold: reduction merization. of the high polymerization temperature and increased Microtomy of the blank blocks of the modified resin intensity of immunogold labeling. was used to choose which resin modification produces The addition of water to the epoxy resin is modest in the best consistency for sectioning. The addition of wa- comparison to the acrylic resins. LR White can absorb

*

MODIFIED QUETOL RESIN FOR IMMUNOCYTOCHEMISTRY

Fig. 1. a: Sample embedded with standard protocol showing good resolution and contrast obtained with the epoxy resin Quetol ( x 22,000). b The omission of osmium and the lack of post-staining results in a lack of resolution in the experiment sample ( x 1,900).

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TABLE 3. Effect of the embedding protocol on immunolabeling of Phanerochaete chrysosporium

Protocol Control 1' Control ' 2 Experimental3 Control 1' Control 22 Experimental3 Control 1' Control 2' Experimental3

Antibody Exocellulase Ligninase Xylanase

No. particles/pm2 0.77 f 0.05 1.69 2 0.26 3.502 0.19 5.32 2 0.24 7.48 f 0.81 20.51 f 2.63 13.95? 0.67 17.11 ? 0.82 25.41 ? 2.76

Labeling increase, % + 119 + 354 -

+ 40

+ 286 + 22 + 82

'74"C, with osmium. 214"C, no osmium. 348"C.no osmium.

12% water (Newman et al., 19831, and Lowicryl can absorb 10% (Bendayan et al., 1987). The maximum water that can be incorporated into Quetol to obtain usable block is 2%. However, the polymerization temperature is decreased by 30°C. The experimental sample was never totally dehydrated. The final dehydration step of sample processing uses either 95% acetone (as in this study) or ethanol. Specimens are embedded with a 1/1ratio of 95% acetone and Quetol (without water) leaving 2.5% water in the solution. The final water concentration is determined by the amount of water added to the modified Quetol. The curing temperature of 48°C of modified Quetol introduces an epoxy resin within the acceptable heat polymerization range for immunocytochemistry study. Quetol can be polymerized within a wide range of curing temperatures, at 90°C for 90 min (Abad et al., 1988; Kushida, 1974) a t 74°C for 8 hr (Abad et al., 1988), and with the addition of water from 74°C to 45°C for 12-36 hr. The temperature can be decreased t o 48°C with the addition of 0.2% water. The resin is suitable for routine TEM with a fast polymerization a t 90°C and immunocytochemistry with a lower curing temperature of 48°C. The lack of resolution of the experimental sample (no osmium) was accentuated since no post-staining of the sample was performed. If the labeling is adequate with an osmicated sample, it represents the best possible result with good preservation and labeling. Removal of surface osmium can be accomplished with sodium metaperiodate (Bendayan and Zollinger, 1983) and an increase of labeling can occur. The sample will retain adequate resolution after treatment. The goal of the investigation should be to determine the labeling intensity required for conclusion. In cases where osmium destroys the antigens, unosmicated samples can be embedded in Quetol with water and the sections poststained. The specimen should have enough resolution and contrast for observation and photography. A substantial increase in labeling intensity was obtained with the addition of water to Quetol. The diameter of hyphae in each treatment was not altered and the increase in labeling was not due to a decrease in surface area. Rat pancreatic tissues embedded in Lowicryl with 10% water showed also no increase in the surface area of organelles (Bendayan et al., 1987). The

sections of modified Quetol expanded on contact with the water in the diamond knife boat. In contrast, sections of unmodified Quetol are not expandable even with chloroform vapor applied to them while floating on the water surface of the diamond knife boat. The expansion of the sections indicates a reduction of the heavily cross-linked bonding of cured modified resin. The reduction of the linkage may reduce the hydrophobicity of the resin and result in a lower background labeling. The decrease of cross-linkage may also increase the relief of the cut surface exposing more antigens to the antibody (Kellenberger et al., 1987). The decrease in the curing temperature from the standard 78°C to 48°C may be the most important factor to introduce modified Quetol as an option for immunocytochemistry. If it has been previously determined that the antigen is not destroyed by treatment with osmium, the sample may be fixed with osmium, cured a t 48"C, etched with sodium metaperiodate prior to labeling, and post-stained after labeling. This protocol takes advantage of the epoxy resin's ability to provide good resolution and contrast (Abad et al., 1988; Kushida, 19741,while reducing the curing temperature to allow the protein antigen to survive polymerization. In this study, a substantial increase in labeling intensity of the experimental treatment was observed. The increase in labeling intensity of the experimental sample is due to the lack of osmium staining and the reduction of curing temperature. The increase in labeling due t o the lack of osmium between the control 2 treatment (no osmium, 74°C) and control 1 treatment (osmium, 74°C) is in agreement with the results of other investigators (Bendayan, 1984b; Bendayan and Zollinger, 1983). The results obtained seem to indicate that the reduction of the polymerization temperature is the major improvement offered by modified Quetol. Osmication of the specimen was omitted in order to test the resolution and contrast of the sample embedded in the modified resin under the least favorable conditions. Under these conditions, the ultrastructure of the specimen is poorly defined. Since osmication increases sample staining, protocols which include treatment with osmium are preferable. Fixation with osmium, followed by etching prior to the immunocytochemistry of the sections, would produce better resolution and contrast of the specimen. This modified Quetol resin combines the high resolution characteristic of an epoxy resin with a medium range polymerization temperature, a combination which is not provided by other epoxy resin formulation. Modified Quetol offers a new resin option for immunocytochemistry study.

ACKNOWLEDGMENTS Published as paper No. 18,203 of the contribution series of the Minnesota Agricultural Experiment Station based on research conducted under Project 22-69H. The author would like to express his gratitude to Dr. Leathers and Dr. Farrell for the xylanase and H8 lignin peroxidase antibodies, respectively, to Dr. Lovrien for CBHI antigens, to Marge Eerdmans for the ELISA testing of the antibodies, to Laura Todd and Dr. Robert

MODIFIED QUETOL RESIN FOR IMMUNOCYTOCHEMISTRY

Fig. 2. a,b An example in the difference in labeling intensity obtained with the ligninase antibody between control 1 and the experimental sample, respectively ( x 32,000 and x 17,000).

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Blanchette for review of the manuscript, and to Meg Clemens for typing the paper.