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Antigen Retrieval in Formaldehyde-Fixed Human Brain Tissue
METHODS: A Companion to Methods in Enzymology 15, 133–140 (1998) Article No. ME980616
Antigen Retrieval in Formaldehyde-Fixed Human Brain Tissue P. Evers,* H. B. M. Uylings,* and A. J. H. Suurmeijer† *Graduate School for Neurosciences Amsterdam, Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands; and †Pathology Laboratory, Martini Hospital, POB 30033, 9700 RM Groningen, The Netherlands
Microwave-stimulated antigen retrieval has become a widely accepted method in both pathology and research laboratories. Since the introduction of the method in 1991, many groups have tried to optimize and standardize it. This review describes the present state of the art. A standard method for microwave-stimulated antigen retrieval in formaldehydefixed paraffin-embedded and nonembedded tissue is presented that results, in general, in very good staining for antibodies used in neuroscience. However, there are still a few antigens that are retrieved not at all or not in an optimal manner. Factors of importance for microwave antigen retrieval are the pH of the retrieval solution and, related to the pH, the temperature and duration of heating. These factors are discussed. q 1998 Academic Press
Since the end of the 19th century, human tissue specimens, including human brain, have usually been fixed by immersion in a formaldehyde solution for economical and practical reasons. Unfortunately, this type of fixation is not optimal for many (immunohistochemical) staining procedures (1). Moreover, the procedure of fixation in a formaldehyde solution is not standardized. This is due mainly to variations in specimen size and duration of fixation. Specimens may vary from 1 to 2 mm for brain biopsies (e.g., tumor biopsies) to several centimeters for whole brains. In neuropathology, fixation time may vary from less than a day for small biopsies to several weeks for whole brains. In neuroscience research, human brain tissue is difficult to obtain, and some-
times only tissue that has been stored in formaldehyde for several years is available. The chemistry of formaldehyde fixation is complex (2–4). In aqueous solution, most formaldehyde exists in its hydrated form, i.e., methylene glycol. Because this chemical equilibrium is in favor of methylene glycol, formaldehyde penetrates rapidly (approximately 1 mm per hour) into the tissue, but it binds and reacts very slowly. Maximal binding of formaldehyde occurs in approximately 1 day and completion of fixation at room temperature takes several days. Tissue fixation with formaldehyde is achieved by its reaction with thiol groups and amino groups of proteins in a crosslinking fashion. The binding of formaldehyde with amino groups is maximal at alkaline pH. However, the formation of secondary crosslinks has an acidic pH optimum. Thus, the amount of crosslinking depends on the fixation time as well as pH, temperature, and concentration of the fixative (5, 6). It is assumed that extensive crosslinking alters the tertiary and quarternary protein structure, which may cause steric barriers that limit the accessibility of the antigen for the antibody. This holds especially for monoclonal antibodies recognizing single epitopes. To overcome the problem of nonreactive antibodies and false-negative immunostaining, various unmasking or retrieval methods are used, e.g., pretreatment with protease (7), formic acid (8), periodic acid (9), guanidine HCl (10), Lugol’s iodine (11), and ultrasound (12). These procedures have been tested or found to be effective with only a fraction of diagnostically useful antigens. Moreover, extensive washing in water or Tris buffer restores immunore133
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activity to some extent, meaning that at least some reactions of formaldehyde with tissue proteins are unstable and reversible (13, 14). Heating tissue in an aqueous medium has proven to be an important factor in antigen unmasking. For this purpose a conventional oven (15), pressure cooker (16), autoclave (17), or microwave oven (15) can be used. Studies comparing these instruments have revealed that microwave heating is a good laboratory tool for unmasking the majority of antigens (18–22).
SHORT REVIEW OF MICROWAVE ANTIGEN RETRIEVAL Paraffin-Embedded Tissue In 1991, Shi et al. (15) reported that microwave heating of paraffin-embedded tissue sections in salt solution prior to immunostaining improved immunoreactivity. With their method, which they called antigen retrieval, high temperature often had a beneficial instead of a deleterious effect on tissue antigenicity. Fifty-two antibodies were tested. Enhanced immunostaining was seen with 39 antibodies, of which 9 showed no difference and 4 showed reduced staining. Distilled water, saturated lead thiocyanate, and zinc sulfate were tested as antigen retrieval solutions. For keratins and vimentin it was found that the sensitivity of immunostaining increased using distilled water, although the best results were obtained with a saturated lead thiocyanate solution. Moreover, these formaldehyde-sensitive antigens could be detected with microwavestimulated antigen retrieval in tissue stored in formaldehyde for 2 years, whereas trypsin digestion failed to restore keratin immunoreacivity. Momose et al. (23) were not able to confirm these promising results. They compared Shi and colleagues’ method with protease treatment using tissue samples fixed overnight and tissue samples fixed for 5 days in neutral buffered formaldehyde. Only 2 of the 17 antibodies tested [including 12 antibodies tested by Shi et al. (15)] clearly profited from antigen retrieval with lead thiocyanate, i.e., glial fibrillary acidic protein (GFAP) and vimentin. The improvement seen in GFAP staining was also found after Pronase (i.e., protease type IV) and trypsin (i.e., protease type III) digestion. Only vimentin was exclusively enhanced by microwave antigen retrieval in the lead thiocyanate solution. The initial report by Shi et al. (15) stimulated Shi and other scientists to modify and
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optimize antigen retrieval in formaldehyde-fixed and paraffin-embedded tissue. The major drawback of Shi and colleagues’ original method was the use of toxic lead thiocyanate. Several authors have described different microwave methods, using less toxic metal and buffer solutions, to retrieve all kinds of antigens (18, 24–34). In an extensive study, Cattoretti et al. (18) found that 110 of 255 antibodies (43%) showed increased immunoreactivity with microwave antigen retrieval using a 0.01 M salt solution at pH 6.0. For 25 of these antibodies, which were considered to be applicable only to frozen sections, microwave heating completely restored reactivity in paraffin-embedded tissue. For 53 other antibodies immunoreactivity was strongly enhanced, and in 32 cases the antigens could be unmasked by microwave pretreatment as well as by protease digestion. The microwave antigen retrieval, however, gave superior results, particularly in tissue fixed for more than 18 h. These authors had the opinion that the molarity of the retrieval solution is an important factor. It appeared that some antigens (including all intermediate filament proteins) depend on strong denaturation conditions (heating in high molarity solutions like 6 M urea and 0.3 M aluminum chloride), whereas other antigens require fine tuning in molarity and salt composition. The good immunostaining results Cattoretti et al. (18) obtained with 0.01 M citrate buffer, pH 6, for a broad range of antigens were confirmed by other groups. In 1995, Shi et al. (35) found that the pH of the retrieval solution is an important factor. Most of the antibodies they examined gave good staining results when Tris–HCl with a high pH (pH 8–9) was used as the retrieval solution. This confirmed the results of Beckstead (24) in 1994 who described that for a number of antibodies the staining largely improved using a Tris–HCl buffer of pH 10. Nonembedded Tissue In 1994, Evers and Uylings (30) applied microwave-stimulated antigen retrieval to free-floating vibratome sections of brain tissue stored in formaldehyde for as long as 10 months. They examined four different retrieval solutions, i.e., distilled water, saturated lead thiocyanate, zinc sulfate, and aluminum chloride, and five antibodies frequently used in neuroscience, i.e., parvalbumin, calbindin D28-K, MAP-2, MAP-5, and SMI-32 (an antibody against the nonphosphorylated part of the neurofilament). Microwave heating led to severe wrinkling of the freefloating 95-mm-thick vibratome sections. Although it
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was not possible to obtain homogenous immunostaining in these wrinkled and folded sections, the results were promising. To overcome the problem of the wrinkled sections they explored whether it was possible to apply the microwave technique to thick tissue slices (about 5 mm thick) before vibratome cutting. Initially, an aluminum chloride solution gave the best results, showing strongly enhanced immunostaining with MAP-2 and SMI-32 antibodies, slightly enhanced immunostaining with parvalbumin, and no difference with the calbindin and MAP-5 antibodies. Shortly afterward in 1994, they modified this technique by using a 0.05 M citrate buffer and showed that different antigens have different optimal pH values. Moreover optimal temperature and irradiation time can be different for different antigens (31). When different antigens have to be detected in the same tissue slice, these different optimal retrieval conditions are very inconvenient. Evers and Uylings solved this problem in general by antigen retrieval in Tris-buffered saline (TBS) with a high pH (pH 9–9.5). This retrieval method gave optimal results with the antibodies examined, i.e., MAP-2, SMI-32, SMI-311, SMI-312, calbindin, parvalbumin, calretinin, and neuropeptide-Y (36).
DESCRIPTION OF METHOD Standard Microwave Antigen Retrieval Protocol Paraffin-Embedded Tissue 1. Attach the paraffin sections to slides coated with an adhesive. Good adhesives are 3-aminopropyltriethoxysilane and poly-L-lysine (Sigma, St. Louis, MO). The use of Superfrost-plus slides (Menzel-gla¨ser, Braunschweich, Germany), slides with a positive charge, is also advisable. 2. After deparaffination and hydration, place the slides in rectangular plastic (TPX) staining jars (Kartell, type Hellendahl) containing approximately 75 ml antigen retrieval solution. In general, Tris– HCl at pH 9 is recommended (35). These jars can hold up to 16 slides. 3. Cover the jars with loose-fitting caps and heat in the center of a household microwave oven with an output of at least 700 W, until boiling. To prevent boiling over and excessive evaporation at higher power levels, it is advisable to irradiate at least two jars at the same time. One jar can serve as a water load. The retrieval solution is kept boiling for two cycles of 5 min. Between these boiling cycles the
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amount of evaporated retrieval solution can be replenished. The time interval between the two boiling cycles is at least 1 min. 4. After heating, remove the staining jars from the microwave oven and allow them to cool for at least 15 min. 5. Rinse the slides twice in distilled water and in phosphate-buffered saline (PBS) for 5 min. 6. Proceed with the immunohistochemical staining procedure. Nonembedded Tissue Slices before Vibratome Sectioning 1. Cut a tissue slice approximately 5 mm thick from the region of interest. 2. Rinse the slice with water (change regularly) for several hours to wash away the formaldehyde fixative. 3. Immerse the slice overnight in the antigen retrieval solution [Evers and Uylings (36) recommended TBS, pH 9.0]. 4. The following morning, place the slice in a plastic jar containing approximately 200 ml of retrieval solution and place the jar in a microwave oven for 10–15 min at full power (700 W), divided into two cycles of 5 or 7.5 min to check the fluid level (Evers and Uylings use a household microwave oven, Miele Electronics 696). 5. After heating, remove the jar from the oven and allow it to cool for 15 min. 6. Rinse the slice in TBS, pH 7.6. 7. Start cutting vibratome sections. 8. Proceed with the immunohistochemical staining procedure. Important Factors Influencing Antigen Retrieval Microwaves Microwaves are electromagnetic waves that can penetrate into material. The penetration depth of microwaves depends on the electric conductivity and physicochemical composition of the material. In human (brain) tissue microwaves penetrate to a depth of about 2 cm; in distilled water at room temperature, to about 3 cm; and in an aqueous 0.3 M NaCl solution, to about 1 cm (37, pp. 42–43). In the oscillating electric fields produced by microwave irradiation, small bipolar molecules such as water are forced to rotate, and this kinetic movement produces instantaneous heat in the material being irradiated. Microwave photons have insufficient energy to break even the weakest molecular bonds. Therefore, all microwave effects are temperature effects. Heat is the
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principal factor determining antigen retrieval outcome. Other sources of heat, e.g., an autoclave, pressure cooker, conventional oven, or heating by steam, have also been applied successfully. The advantage of a microwave oven is its production of heat in a quick and reproducible way. All data on size, volume, and shape of the specimen, concentration (molarity) of the retrieval solution, position in the oven, and temperature are controllable and can be reported [see, e.g., (38)]. Intensity and Duration of Heating In the original standard microwave method described by Shi et al. (15) two boiling cycles of 5 min are applied. With three 50-ml Coplin jars in a microwave oven and a power of 700 W, the boiling point is reached in 140–145 s. So, net boiling time is less than 8 min. The same thing occurs when two 75-ml Hellendahl staining jars are used. Here, one should bear in mind that the heating performance of different types of microwave ovens may vary considerably at a certain power level because of hot spots in the oven. This occurs in particular with different shapes of containers and small volumes, which leads to variation in the efficiency of antigen retrieval. For standardization of the heating condition during irradiation, monitoring of the temperature is required. In this respect the calibration method of Tacha and Chen (39) might be helpful. The net power P of a microwave oven can be determined with the simple procedure described by Kok and Boon (37, pp. 63– 64) or the guideline issued by the International Microwave Power Institute (37, p. 406). Several studies have shown that some epitopes may require controlled boiling for longer periods. Munakata and Hendricks (27) studied the effect of fixation time and microwave heating time on the retrieval of Ki-67 with monoclonal antibody MIB-1 in citrate buffer, pH 6. Poor immunostaining was observed in tissue fixed for 48 h, unless a heating time of at least 14 min was used (boiling time was about 12 min). Von Wasielewski et al. (40) reported that maximal enhancement of staining was achieved by continuous boiling of slides for 20–35 min in citrate buffer, pH 6.0, whereas a reduced net boiling time of 7 min led to suboptimal enhancement. These authors also compared different fixation times. With fixation in
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4% buffered formaldehyde for 24 h enhanced staining was observed in 9 of 52 antibodies (17%). If formaldehyde fixation was extended to 6 weeks, staining improved to 26 of 52 antibodies (50%). These data clearly indicate that the degree of antigen retrieval is proportional to the duration of heating and also depends on fixation conditions. Since in daily practice the length of formaldehyde fixation may vary from a few hours to several days, or even several months, 10–20 min of heat exposure provides a good safeguard. These findings are all based on paraffin-embedded sections that had been brought to the boil in a microwave oven. Other groups (19, 22, 26) describe that retrieval at the lower temperature of 607C enhances immunostaining when the duration of heating is prolonged several hours. However, heating at 907C or boiling generally gives better immunostaining, while morphologic detail is still well preserved. Evers and Uylings (31), in their study on antigen retrieval of nonembedded tissue, reported that high temperatures (ú857C) are necessary, but that different antibodies require different temperatures and different irradiation times related to the pH of the antigen retrieval solution to obtain optimal results: 2 hours at 907C for SMI-32 (non-phosphorylated neurofilament) at pH 2.5 and 10 min of boiling for the MAP2 antibody at pH 4.5. Moreover, it was noted that both antibodies require another optimal pH of the retrieval solution. The influence of the pH of the retrieval solution appears to be decisive, as discussed below. During the 15-min cooling phase the temperature of the retrieval solution decreases to approximately 507C. Actually, the sections are left standing in a hot solution for some minutes, which may explain the experience of some authors that the retrieval of some antigens is less effective if this cooling step is omitted after 10 min of microwave heating. However, for the large majority of antigens the cooling down period is not essential for retrieval. The main purpose of the cooling down step is to prevent detachment of tissue sections from the slide. pH of the Retrieval Solution Since 1994, the importance of the pH of the retrieval solution has been noted. Evers and Uylings (31) found that optimal pretreatment for antigen re-
FIG. 1. Vibratome sections of human cortical brain tissue, fixed for 4 years in formaldehyde, stained after microwave pretreatment for (a) parvalbumin, (b) calretinin, (c) SMI-311, and (d) SMI-32. Without pretreatment it was not possible to obtain any staining except for some vaguely stained parvalbumin-positive somata. Bar, 100 mm.
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FIG. 2. Vibratome sections of human cortical brain tissue, fixed for 4 years in formaldehyde, stained after microwave pretreatment for (a) parvalbumin, (b) calbindin, (c) SMI-311, and (d) SMI-32. Without pretreatment it was not possible to obtain any staining with the SMI-32 and SMI-311 antibodies and only some vaguely stained somata with the parvalbumin and calbindin antibodies. Bar, 50 mm.
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trieval includes adjustment of the pH value of the retrieval solution, irradiation time, and also temperature. In their study they tested pH ranging from 1.0 to 6.0. Within this pH range they had to apply different retrieval solutions to nonembedded tissue slices. The importance of the pH of the retrieval solution was also noted by Beckstead (24) and Shi et al. (35). Shi et al. (35) compared different buffer solutions with pH ranging from 1 to 10 and evaluated monoclonal antibodies to cytoplasmic antigens (AE1, HMB-45, NSE), nuclear antigens (MIB-1, PCNA, ER), and cell surface antigens (MT1, L26, EMA) on formaldehyde-fixed, paraffin-embedded tissue. Three patterns of pH effects on antigen retrieval immunostaining were found: a type A pattern (for AE1, NSE, PCNA, L26 and EMA) with excellent retrieval throughout the entire pH range tested; a type B pattern (for MIB-1 and ER) with strong immunostaining at very low and high pH and a strong decrease in staining intensity at pH 3–6; and a type C pattern (for HMB-45 and MT1) with increasing staining intensity in proportion to increasing pH, but weak staining at low pH. The chemical composition of the retrieval solution appeared to be less important than the pH. Tris–HCl buffer was preferred because it was not necessary to add NaOH to obtain the high pH of 9. The disadvantage of adding NaOH to the retrieval solution is that sections tend to detach from the slides during microwaving. Evers and Uylings (36) also investigated the influence of high pH of the retrieval solution on their method of antigen retrieval in nonembedded tissue slices. This was important for the development of a method allowing different immunostainings on vibratome sections of the same tissue slice. The antibodies tested were SMI-32 and SMI-311 (both against nonphosphorylated epitopes of the neurofilament), SMI-312 (against the phosphorylated part of the neurofilament), MAP-2 (microtubule-associated protein), calbindin D-28K, parvalbumin and calretinin (calcium-binding proteins), and neuropeptide-Y (NPY). SMI-32 and MAP-2 are known to result in optimal retrieval if an antigen retrieval solution with different low pH is used (2.5 and 4.5, respectively). Until this study the antigen retrieval of calcium-binding proteins was very poor after pretreatment with low-pH solutions, and NPY was believed to be unaffected by prolonged storage in formaldehyde (41, 42). The conclusion was drawn that a TBS solution of pH 9–9.5 gives excellent staining results, in tissue fixed for years and with all antibodies. Even the intensity of immunostained NPY fibers was con-
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siderably higher than without antigen retrieval. It also appeared that addition of NaOH to the retrieval solution has to be avoided because the tissue slices become very soft, which makes it difficult to cut vibratome sections afterward. Examples of immunohistochemical staining on vibratome sections after microwave pretreatment in a TBS solution at pH 9.0 are shown in Figs. 1 and 2.
CONCLUDING REMARKS Microwave-stimulated antigen retrieval has proved to be an important tool in the retrieval of many antigens. The introduction of this technique has made it possible to use immunohistochemical staining methods on (human) brain tissue, which before were considered useless for this purpose because of the (too) long period of fixation. The methods can be applied to paraffin and celloidin (22) sections, but also to nonembedded tissue from which vibratome sections are prepared after retrieval. The mechanism of antigen retrieval is poorly understood and by trial and error the technique has been optimized for its use with many antibodies. Beckstead (24), Shi et al. (35), and Evers and Uylings (36) took an important step in finding a standard optimal method to retrieve all kinds of antigens. The most important factor involved in microwave-stimulated antigen retrieval seems to be the pH of the retrieval solution in combination with the temperature. For many antibodies microwave boiling of tissue for 10 min in Tris buffer at pH 9.0 seems most appropriate. However, some antibodies may require other optimal retrieval conditions, which can be determined by varying pH, temperature, time, and retrieval solution. Standardization of antigen retrieval is, in particular, essential for quantitative immunohistochemistry, e.g., with proliferation markers and hormone receptor antibodies. Our test battery, as the one suggested by Shi et al. (22), may be of great help. This test compares three temperature levels (mid-high, high, and superhigh, i.e., 807C, 907C, and boiling) in combination with three pH values (low pH, 2.5; intermediate pH, 6; high pH, 9). One should keep in mind that lower temperatures require longer irradiation times (22, 31, 36). The increased sensitivity of immunostaining with antigen retrieval may warrant adjustment of the dilution of the primary and/or secondary antibody in the staining protocol. This also reduces the costs of staining. False-positive immunostaining has been
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observed in a few cases with antigen retrieval at low pH. Therefore one should always review a negative control slide (omission of the first antibody step) and compare staining in paraffin or vibratome sections with staining in positive control sections (i.e., frozen sections). It is also advisable to run some specificity control tests, e.g., incubation of control sections in antiserum after preadsorption with the protein (42). Although antigen retrieval is now used worldwide with great satisfaction, there is definitely a need for further studies on optimization and possibly standardization of the technique.
ACKNOWLEDGMENTS The authors thank Mr. G. van der Meulen for his photographical assistance and Ms. O. Pach for correcting the English.
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16. Miller, K., Auld, J., Jessup, E., Rhodes, A., and Ashton Key, M. (1995) Appl. Immunohistochem. 1, 60–64. 17. Shin, R-W, Iwaki, T., Kitamoto, T., and Tateishi, J. (1991) Lab. Invest. 64, 693–702. 18. Cattoretti, G., Pileri, S., Parravicini, C., Becker, M. H. G., Poggi, S., Bifulco, C., Key, G., D’Amato, L., Sabattini, E., Feudale, E., Reynolds, F., Gerdes, J., and Rilke, F. (1993) J. Pathol. 171, 83–98. 19. Igarashi, H., Sugimura, H., Maruyama, K., Kitayama, Y., Ohta, I., Suzuki M., Tanaka, M., Dobashi, Y., and Kino, I. (1994) APMIS 102, 295–307. 20. Sherriff, F. E., Bridges, L. R., and Jackson, P. (1994) Neuroreport 5, 1085–1088. 21. Taylor, C. R., Shi, S-R., and Cote, R. J. (1996) Appl. Immunohistochem. 4, 144–166. 22. Shi, S-R., Cote, R. J., and Taylor, C. R. (1997) J. Histochem. Cytochem. 45, 327–343. 23. Momose, H., Metha, P., and Battifora, H. (1993) Appl. Immunohistochem. 1, 77–82. 24. Beckstead, J. H. (1994) Appl. Immunohistochem. 2, 274–281. 25. Cattoretti, G., and Suurmeijer, A. J. H. (1995) Adv. Anat. Pathol. 2, 2–9. 26. Lucassen, P. J., Ravid, R., Gonatas, N. K., and Swaab, D. F. (1993) Brain Res. 632, 105–113. 27. Munakata, S., and Hendricks, J. B. (1993) J. Histochem. Cytochem. 41, 1241–1246. 28. Suurmeijer, A. J. H., and Boon, M. E. (1993) Appl. Immunohistochem. 1, 143–148. 29. Ainley, C. D., and Ironside, J. W. (1994) J. Neurosci. Methods 55, 183–190. 30. Evers, P., and Uylings, H. B. M. (1994) J. Neurosci. Methods 55, 163–172. 31. Evers, P., and Uylings, H. B. M. (1994) J. Histochem. Cytochem. 42, 1555–1563. 32. Shi, S-R., Chaiwun, B., Young, L., Imam, A., Cote, R. J., and Taylor, C. R. (1994) Biotechn. Histochem. 69, 213–215. 33. Yachnis, A. T., and Trojanowski, J. Q. (1994) J. Neurosci. Methods 55, 191–200. 34. Stra¨ter, J., Gu¨nthert, A. R., Bru¨derlein, S., and Mo¨ller, P. (1995) Histochemistry 103, 157–160. 35. Shi, S-R., Imam, A., Young, L., Cote, R. J., and Taylor, C. R. (1995) J. Histochem. Cytochem. 43, 193–201. 36. Evers, P., and Uylings, H. B. M. (1997) J. Neurosci. Methods 72, 197–207. 37. Kok, L. P., and Boon, M. E. (1992) Microwave Cookbook for Microscopists: Art and Science of Visualization, pp. 42–43, Coulomb Press Leyden, Leiden. 38. Marani, E. (1991) Eur. J. Morphol. 29, 181. 39. Tacha, D. E., and Chen, T. (1994) J. Histotechnol. 17, 365– 366. 40. Von Wasielewski, R., Werner, M., Nolte, M., Wilkens, L., and Georgii, A. (1994) Histochemistry 102, 165–172. 41. Uylings, H. B. M., and Delalle, I. (1997) J. Comp. Neurol. 379, 523–540. 42. Delalle, I., Evers, P., Kostovic´, I., and Uylings, H. B. M. (1997) J. Comp. Neurol. 379, 515–522.
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Antigen Retrieval in Formaldehyde-Fixed Human Brain Tissue P. Evers,* H. B. M. Uylings,* and A. J. H. Suurmeijer† *Graduate School for Neurosciences Amsterdam, Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands; and †Pathology Laboratory, Martini Hospital, POB 30033, 9700 RM Groningen, The Netherlands
Microwave-stimulated antigen retrieval has become a widely accepted method in both pathology and research laboratories. Since the introduction of the method in 1991, many groups have tried to optimize and standardize it. This review describes the present state of the art. A standard method for microwave-stimulated antigen retrieval in formaldehydefixed paraffin-embedded and nonembedded tissue is presented that results, in general, in very good staining for antibodies used in neuroscience. However, there are still a few antigens that are retrieved not at all or not in an optimal manner. Factors of importance for microwave antigen retrieval are the pH of the retrieval solution and, related to the pH, the temperature and duration of heating. These factors are discussed. q 1998 Academic Press
Since the end of the 19th century, human tissue specimens, including human brain, have usually been fixed by immersion in a formaldehyde solution for economical and practical reasons. Unfortunately, this type of fixation is not optimal for many (immunohistochemical) staining procedures (1). Moreover, the procedure of fixation in a formaldehyde solution is not standardized. This is due mainly to variations in specimen size and duration of fixation. Specimens may vary from 1 to 2 mm for brain biopsies (e.g., tumor biopsies) to several centimeters for whole brains. In neuropathology, fixation time may vary from less than a day for small biopsies to several weeks for whole brains. In neuroscience research, human brain tissue is difficult to obtain, and some-
times only tissue that has been stored in formaldehyde for several years is available. The chemistry of formaldehyde fixation is complex (2–4). In aqueous solution, most formaldehyde exists in its hydrated form, i.e., methylene glycol. Because this chemical equilibrium is in favor of methylene glycol, formaldehyde penetrates rapidly (approximately 1 mm per hour) into the tissue, but it binds and reacts very slowly. Maximal binding of formaldehyde occurs in approximately 1 day and completion of fixation at room temperature takes several days. Tissue fixation with formaldehyde is achieved by its reaction with thiol groups and amino groups of proteins in a crosslinking fashion. The binding of formaldehyde with amino groups is maximal at alkaline pH. However, the formation of secondary crosslinks has an acidic pH optimum. Thus, the amount of crosslinking depends on the fixation time as well as pH, temperature, and concentration of the fixative (5, 6). It is assumed that extensive crosslinking alters the tertiary and quarternary protein structure, which may cause steric barriers that limit the accessibility of the antigen for the antibody. This holds especially for monoclonal antibodies recognizing single epitopes. To overcome the problem of nonreactive antibodies and false-negative immunostaining, various unmasking or retrieval methods are used, e.g., pretreatment with protease (7), formic acid (8), periodic acid (9), guanidine HCl (10), Lugol’s iodine (11), and ultrasound (12). These procedures have been tested or found to be effective with only a fraction of diagnostically useful antigens. Moreover, extensive washing in water or Tris buffer restores immunore133
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activity to some extent, meaning that at least some reactions of formaldehyde with tissue proteins are unstable and reversible (13, 14). Heating tissue in an aqueous medium has proven to be an important factor in antigen unmasking. For this purpose a conventional oven (15), pressure cooker (16), autoclave (17), or microwave oven (15) can be used. Studies comparing these instruments have revealed that microwave heating is a good laboratory tool for unmasking the majority of antigens (18–22).
SHORT REVIEW OF MICROWAVE ANTIGEN RETRIEVAL Paraffin-Embedded Tissue In 1991, Shi et al. (15) reported that microwave heating of paraffin-embedded tissue sections in salt solution prior to immunostaining improved immunoreactivity. With their method, which they called antigen retrieval, high temperature often had a beneficial instead of a deleterious effect on tissue antigenicity. Fifty-two antibodies were tested. Enhanced immunostaining was seen with 39 antibodies, of which 9 showed no difference and 4 showed reduced staining. Distilled water, saturated lead thiocyanate, and zinc sulfate were tested as antigen retrieval solutions. For keratins and vimentin it was found that the sensitivity of immunostaining increased using distilled water, although the best results were obtained with a saturated lead thiocyanate solution. Moreover, these formaldehyde-sensitive antigens could be detected with microwavestimulated antigen retrieval in tissue stored in formaldehyde for 2 years, whereas trypsin digestion failed to restore keratin immunoreacivity. Momose et al. (23) were not able to confirm these promising results. They compared Shi and colleagues’ method with protease treatment using tissue samples fixed overnight and tissue samples fixed for 5 days in neutral buffered formaldehyde. Only 2 of the 17 antibodies tested [including 12 antibodies tested by Shi et al. (15)] clearly profited from antigen retrieval with lead thiocyanate, i.e., glial fibrillary acidic protein (GFAP) and vimentin. The improvement seen in GFAP staining was also found after Pronase (i.e., protease type IV) and trypsin (i.e., protease type III) digestion. Only vimentin was exclusively enhanced by microwave antigen retrieval in the lead thiocyanate solution. The initial report by Shi et al. (15) stimulated Shi and other scientists to modify and
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optimize antigen retrieval in formaldehyde-fixed and paraffin-embedded tissue. The major drawback of Shi and colleagues’ original method was the use of toxic lead thiocyanate. Several authors have described different microwave methods, using less toxic metal and buffer solutions, to retrieve all kinds of antigens (18, 24–34). In an extensive study, Cattoretti et al. (18) found that 110 of 255 antibodies (43%) showed increased immunoreactivity with microwave antigen retrieval using a 0.01 M salt solution at pH 6.0. For 25 of these antibodies, which were considered to be applicable only to frozen sections, microwave heating completely restored reactivity in paraffin-embedded tissue. For 53 other antibodies immunoreactivity was strongly enhanced, and in 32 cases the antigens could be unmasked by microwave pretreatment as well as by protease digestion. The microwave antigen retrieval, however, gave superior results, particularly in tissue fixed for more than 18 h. These authors had the opinion that the molarity of the retrieval solution is an important factor. It appeared that some antigens (including all intermediate filament proteins) depend on strong denaturation conditions (heating in high molarity solutions like 6 M urea and 0.3 M aluminum chloride), whereas other antigens require fine tuning in molarity and salt composition. The good immunostaining results Cattoretti et al. (18) obtained with 0.01 M citrate buffer, pH 6, for a broad range of antigens were confirmed by other groups. In 1995, Shi et al. (35) found that the pH of the retrieval solution is an important factor. Most of the antibodies they examined gave good staining results when Tris–HCl with a high pH (pH 8–9) was used as the retrieval solution. This confirmed the results of Beckstead (24) in 1994 who described that for a number of antibodies the staining largely improved using a Tris–HCl buffer of pH 10. Nonembedded Tissue In 1994, Evers and Uylings (30) applied microwave-stimulated antigen retrieval to free-floating vibratome sections of brain tissue stored in formaldehyde for as long as 10 months. They examined four different retrieval solutions, i.e., distilled water, saturated lead thiocyanate, zinc sulfate, and aluminum chloride, and five antibodies frequently used in neuroscience, i.e., parvalbumin, calbindin D28-K, MAP-2, MAP-5, and SMI-32 (an antibody against the nonphosphorylated part of the neurofilament). Microwave heating led to severe wrinkling of the freefloating 95-mm-thick vibratome sections. Although it
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was not possible to obtain homogenous immunostaining in these wrinkled and folded sections, the results were promising. To overcome the problem of the wrinkled sections they explored whether it was possible to apply the microwave technique to thick tissue slices (about 5 mm thick) before vibratome cutting. Initially, an aluminum chloride solution gave the best results, showing strongly enhanced immunostaining with MAP-2 and SMI-32 antibodies, slightly enhanced immunostaining with parvalbumin, and no difference with the calbindin and MAP-5 antibodies. Shortly afterward in 1994, they modified this technique by using a 0.05 M citrate buffer and showed that different antigens have different optimal pH values. Moreover optimal temperature and irradiation time can be different for different antigens (31). When different antigens have to be detected in the same tissue slice, these different optimal retrieval conditions are very inconvenient. Evers and Uylings solved this problem in general by antigen retrieval in Tris-buffered saline (TBS) with a high pH (pH 9–9.5). This retrieval method gave optimal results with the antibodies examined, i.e., MAP-2, SMI-32, SMI-311, SMI-312, calbindin, parvalbumin, calretinin, and neuropeptide-Y (36).
DESCRIPTION OF METHOD Standard Microwave Antigen Retrieval Protocol Paraffin-Embedded Tissue 1. Attach the paraffin sections to slides coated with an adhesive. Good adhesives are 3-aminopropyltriethoxysilane and poly-L-lysine (Sigma, St. Louis, MO). The use of Superfrost-plus slides (Menzel-gla¨ser, Braunschweich, Germany), slides with a positive charge, is also advisable. 2. After deparaffination and hydration, place the slides in rectangular plastic (TPX) staining jars (Kartell, type Hellendahl) containing approximately 75 ml antigen retrieval solution. In general, Tris– HCl at pH 9 is recommended (35). These jars can hold up to 16 slides. 3. Cover the jars with loose-fitting caps and heat in the center of a household microwave oven with an output of at least 700 W, until boiling. To prevent boiling over and excessive evaporation at higher power levels, it is advisable to irradiate at least two jars at the same time. One jar can serve as a water load. The retrieval solution is kept boiling for two cycles of 5 min. Between these boiling cycles the
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amount of evaporated retrieval solution can be replenished. The time interval between the two boiling cycles is at least 1 min. 4. After heating, remove the staining jars from the microwave oven and allow them to cool for at least 15 min. 5. Rinse the slides twice in distilled water and in phosphate-buffered saline (PBS) for 5 min. 6. Proceed with the immunohistochemical staining procedure. Nonembedded Tissue Slices before Vibratome Sectioning 1. Cut a tissue slice approximately 5 mm thick from the region of interest. 2. Rinse the slice with water (change regularly) for several hours to wash away the formaldehyde fixative. 3. Immerse the slice overnight in the antigen retrieval solution [Evers and Uylings (36) recommended TBS, pH 9.0]. 4. The following morning, place the slice in a plastic jar containing approximately 200 ml of retrieval solution and place the jar in a microwave oven for 10–15 min at full power (700 W), divided into two cycles of 5 or 7.5 min to check the fluid level (Evers and Uylings use a household microwave oven, Miele Electronics 696). 5. After heating, remove the jar from the oven and allow it to cool for 15 min. 6. Rinse the slice in TBS, pH 7.6. 7. Start cutting vibratome sections. 8. Proceed with the immunohistochemical staining procedure. Important Factors Influencing Antigen Retrieval Microwaves Microwaves are electromagnetic waves that can penetrate into material. The penetration depth of microwaves depends on the electric conductivity and physicochemical composition of the material. In human (brain) tissue microwaves penetrate to a depth of about 2 cm; in distilled water at room temperature, to about 3 cm; and in an aqueous 0.3 M NaCl solution, to about 1 cm (37, pp. 42–43). In the oscillating electric fields produced by microwave irradiation, small bipolar molecules such as water are forced to rotate, and this kinetic movement produces instantaneous heat in the material being irradiated. Microwave photons have insufficient energy to break even the weakest molecular bonds. Therefore, all microwave effects are temperature effects. Heat is the
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principal factor determining antigen retrieval outcome. Other sources of heat, e.g., an autoclave, pressure cooker, conventional oven, or heating by steam, have also been applied successfully. The advantage of a microwave oven is its production of heat in a quick and reproducible way. All data on size, volume, and shape of the specimen, concentration (molarity) of the retrieval solution, position in the oven, and temperature are controllable and can be reported [see, e.g., (38)]. Intensity and Duration of Heating In the original standard microwave method described by Shi et al. (15) two boiling cycles of 5 min are applied. With three 50-ml Coplin jars in a microwave oven and a power of 700 W, the boiling point is reached in 140–145 s. So, net boiling time is less than 8 min. The same thing occurs when two 75-ml Hellendahl staining jars are used. Here, one should bear in mind that the heating performance of different types of microwave ovens may vary considerably at a certain power level because of hot spots in the oven. This occurs in particular with different shapes of containers and small volumes, which leads to variation in the efficiency of antigen retrieval. For standardization of the heating condition during irradiation, monitoring of the temperature is required. In this respect the calibration method of Tacha and Chen (39) might be helpful. The net power P of a microwave oven can be determined with the simple procedure described by Kok and Boon (37, pp. 63– 64) or the guideline issued by the International Microwave Power Institute (37, p. 406). Several studies have shown that some epitopes may require controlled boiling for longer periods. Munakata and Hendricks (27) studied the effect of fixation time and microwave heating time on the retrieval of Ki-67 with monoclonal antibody MIB-1 in citrate buffer, pH 6. Poor immunostaining was observed in tissue fixed for 48 h, unless a heating time of at least 14 min was used (boiling time was about 12 min). Von Wasielewski et al. (40) reported that maximal enhancement of staining was achieved by continuous boiling of slides for 20–35 min in citrate buffer, pH 6.0, whereas a reduced net boiling time of 7 min led to suboptimal enhancement. These authors also compared different fixation times. With fixation in
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4% buffered formaldehyde for 24 h enhanced staining was observed in 9 of 52 antibodies (17%). If formaldehyde fixation was extended to 6 weeks, staining improved to 26 of 52 antibodies (50%). These data clearly indicate that the degree of antigen retrieval is proportional to the duration of heating and also depends on fixation conditions. Since in daily practice the length of formaldehyde fixation may vary from a few hours to several days, or even several months, 10–20 min of heat exposure provides a good safeguard. These findings are all based on paraffin-embedded sections that had been brought to the boil in a microwave oven. Other groups (19, 22, 26) describe that retrieval at the lower temperature of 607C enhances immunostaining when the duration of heating is prolonged several hours. However, heating at 907C or boiling generally gives better immunostaining, while morphologic detail is still well preserved. Evers and Uylings (31), in their study on antigen retrieval of nonembedded tissue, reported that high temperatures (ú857C) are necessary, but that different antibodies require different temperatures and different irradiation times related to the pH of the antigen retrieval solution to obtain optimal results: 2 hours at 907C for SMI-32 (non-phosphorylated neurofilament) at pH 2.5 and 10 min of boiling for the MAP2 antibody at pH 4.5. Moreover, it was noted that both antibodies require another optimal pH of the retrieval solution. The influence of the pH of the retrieval solution appears to be decisive, as discussed below. During the 15-min cooling phase the temperature of the retrieval solution decreases to approximately 507C. Actually, the sections are left standing in a hot solution for some minutes, which may explain the experience of some authors that the retrieval of some antigens is less effective if this cooling step is omitted after 10 min of microwave heating. However, for the large majority of antigens the cooling down period is not essential for retrieval. The main purpose of the cooling down step is to prevent detachment of tissue sections from the slide. pH of the Retrieval Solution Since 1994, the importance of the pH of the retrieval solution has been noted. Evers and Uylings (31) found that optimal pretreatment for antigen re-
FIG. 1. Vibratome sections of human cortical brain tissue, fixed for 4 years in formaldehyde, stained after microwave pretreatment for (a) parvalbumin, (b) calretinin, (c) SMI-311, and (d) SMI-32. Without pretreatment it was not possible to obtain any staining except for some vaguely stained parvalbumin-positive somata. Bar, 100 mm.
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FIG. 2. Vibratome sections of human cortical brain tissue, fixed for 4 years in formaldehyde, stained after microwave pretreatment for (a) parvalbumin, (b) calbindin, (c) SMI-311, and (d) SMI-32. Without pretreatment it was not possible to obtain any staining with the SMI-32 and SMI-311 antibodies and only some vaguely stained somata with the parvalbumin and calbindin antibodies. Bar, 50 mm.
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trieval includes adjustment of the pH value of the retrieval solution, irradiation time, and also temperature. In their study they tested pH ranging from 1.0 to 6.0. Within this pH range they had to apply different retrieval solutions to nonembedded tissue slices. The importance of the pH of the retrieval solution was also noted by Beckstead (24) and Shi et al. (35). Shi et al. (35) compared different buffer solutions with pH ranging from 1 to 10 and evaluated monoclonal antibodies to cytoplasmic antigens (AE1, HMB-45, NSE), nuclear antigens (MIB-1, PCNA, ER), and cell surface antigens (MT1, L26, EMA) on formaldehyde-fixed, paraffin-embedded tissue. Three patterns of pH effects on antigen retrieval immunostaining were found: a type A pattern (for AE1, NSE, PCNA, L26 and EMA) with excellent retrieval throughout the entire pH range tested; a type B pattern (for MIB-1 and ER) with strong immunostaining at very low and high pH and a strong decrease in staining intensity at pH 3–6; and a type C pattern (for HMB-45 and MT1) with increasing staining intensity in proportion to increasing pH, but weak staining at low pH. The chemical composition of the retrieval solution appeared to be less important than the pH. Tris–HCl buffer was preferred because it was not necessary to add NaOH to obtain the high pH of 9. The disadvantage of adding NaOH to the retrieval solution is that sections tend to detach from the slides during microwaving. Evers and Uylings (36) also investigated the influence of high pH of the retrieval solution on their method of antigen retrieval in nonembedded tissue slices. This was important for the development of a method allowing different immunostainings on vibratome sections of the same tissue slice. The antibodies tested were SMI-32 and SMI-311 (both against nonphosphorylated epitopes of the neurofilament), SMI-312 (against the phosphorylated part of the neurofilament), MAP-2 (microtubule-associated protein), calbindin D-28K, parvalbumin and calretinin (calcium-binding proteins), and neuropeptide-Y (NPY). SMI-32 and MAP-2 are known to result in optimal retrieval if an antigen retrieval solution with different low pH is used (2.5 and 4.5, respectively). Until this study the antigen retrieval of calcium-binding proteins was very poor after pretreatment with low-pH solutions, and NPY was believed to be unaffected by prolonged storage in formaldehyde (41, 42). The conclusion was drawn that a TBS solution of pH 9–9.5 gives excellent staining results, in tissue fixed for years and with all antibodies. Even the intensity of immunostained NPY fibers was con-
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siderably higher than without antigen retrieval. It also appeared that addition of NaOH to the retrieval solution has to be avoided because the tissue slices become very soft, which makes it difficult to cut vibratome sections afterward. Examples of immunohistochemical staining on vibratome sections after microwave pretreatment in a TBS solution at pH 9.0 are shown in Figs. 1 and 2.
CONCLUDING REMARKS Microwave-stimulated antigen retrieval has proved to be an important tool in the retrieval of many antigens. The introduction of this technique has made it possible to use immunohistochemical staining methods on (human) brain tissue, which before were considered useless for this purpose because of the (too) long period of fixation. The methods can be applied to paraffin and celloidin (22) sections, but also to nonembedded tissue from which vibratome sections are prepared after retrieval. The mechanism of antigen retrieval is poorly understood and by trial and error the technique has been optimized for its use with many antibodies. Beckstead (24), Shi et al. (35), and Evers and Uylings (36) took an important step in finding a standard optimal method to retrieve all kinds of antigens. The most important factor involved in microwave-stimulated antigen retrieval seems to be the pH of the retrieval solution in combination with the temperature. For many antibodies microwave boiling of tissue for 10 min in Tris buffer at pH 9.0 seems most appropriate. However, some antibodies may require other optimal retrieval conditions, which can be determined by varying pH, temperature, time, and retrieval solution. Standardization of antigen retrieval is, in particular, essential for quantitative immunohistochemistry, e.g., with proliferation markers and hormone receptor antibodies. Our test battery, as the one suggested by Shi et al. (22), may be of great help. This test compares three temperature levels (mid-high, high, and superhigh, i.e., 807C, 907C, and boiling) in combination with three pH values (low pH, 2.5; intermediate pH, 6; high pH, 9). One should keep in mind that lower temperatures require longer irradiation times (22, 31, 36). The increased sensitivity of immunostaining with antigen retrieval may warrant adjustment of the dilution of the primary and/or secondary antibody in the staining protocol. This also reduces the costs of staining. False-positive immunostaining has been
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observed in a few cases with antigen retrieval at low pH. Therefore one should always review a negative control slide (omission of the first antibody step) and compare staining in paraffin or vibratome sections with staining in positive control sections (i.e., frozen sections). It is also advisable to run some specificity control tests, e.g., incubation of control sections in antiserum after preadsorption with the protein (42). Although antigen retrieval is now used worldwide with great satisfaction, there is definitely a need for further studies on optimization and possibly standardization of the technique.
ACKNOWLEDGMENTS The authors thank Mr. G. van der Meulen for his photographical assistance and Ms. O. Pach for correcting the English.
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