Evaluation of antigen retrieval buffer systems

Journal of Molecular Histology 35: 409 416, 2004. 2004 Kluwer Academic Publishers. Printed in the Netherlands. Evaluation of antigen retrieval buffer systems Seok H. Kim 1,2, Myeong C. Kook 3, Young K. Shin 2,4, Seong H. Park 3 & Hyung G. Song 1,2, 1 Department of Pathology, Chungbuk National University College of Medicine, 62 Kaesin-dong, Cheongju, Chungbuk, South Korea, 361-763 2 DiNonA Inc, Seoul, Korea 3 Department of Pathology, Seoul National University College of Medicine, Seoul, Korea 4 Department of Pharmacy, Seoul National University College of Pharmacy, Seoul, Korea Author for correspondence Received 16 June 2003 and in revised form 8 January 2004 Summary The introduction of antigen retrieval (AR) techniques has dramatically improved the sensitivity of immunohistochemical detection of various antigens in formalin-fixed, paraffin-embedded tissues. The microwave-heating and pressure-cooking procedures are the most effective AR methods reported to date. Although extensive efforts have been made to optimize AR procedures using these two methods, previous studies have not led to a standard protocol applicable to all antibodies derived from different clones. In this study we have investigated the optimal AR buffer conditions for 29 antibodies that are in common use for diagnostic purposes in hospitals worldwide. Borate (ph 8.0) and Tris buffer (ph 9.5) yielded the highest retrieved antigen immunoreactivity against most antibodies as compared to other buffers tested. In addition, the microwave pressure-cooking gave better results than microwave-heating alone. Therefore, borate (ph 8.0) or Tris (ph 9.5) buffer used in conjunction with the pressure-cooking procedure is strongly recommended for standard routine use. Introduction Immunohistochemistry has become an essential tool in both diagnostic pathology and morphology-based research, as a consequence of a series of technical advances applied to routine formalin-fixed tissues. In addition, the development of the antigen retrieval (AR) technique has dramatically improved the applicability of immunohistochemistry for archival tissues (Shi et al. 1991). Heat-induced AR has been reported to be generally superior compared to enzymatic digestion in terms of the sensitivity and reproducibility of immunostaining (Cattoretti et al. 1993). The efficacy of heat-induced AR, however, is known to be influenced by several factors, including the heating conditions and the physicochemical characteristics of the AR buffer (Taylor et al. 1996a,b). The key factors that need to be optimized for a given antibody are the ph and chemical composition of the AR buffer, whereas other factors, such as the duration and the intensity of heating, have relatively little effect (Shi et al. 1995). With some antigens, no significant variation in the efficiency of AR has been observed across the whole ph range from 1.0 to 10.0. On the other hand, many other antigens showed a dramatic decrease in the intensity of immunoreactive staining after pretreatment at a ph between 3.0 and 6.0, but strong staining above and below this critical zone. Moreover, several antigens are unreactive or display weak focal immunoreactive staining after low ph treatment (ph 1 2) but excellent results after pretreatment in the high ph range (Shi et al. 1995). These results suggest that the universal use of citrate buffer (ph 6.0) may not be the best choice for optimal immunostaining in many cases. However, the evaluation of AR buffers that lead to optimal immunostaining by the range of antibodies widely used for routine diagnosis and research has not been adequately studied. Therefore, in this study we tested seven buffers of different ph and chemical composition to find out which gave the optimal immunostaining for 29 commonly used antibodies. Our results demonstrated that pretreatment with borate (ph 8.0) or Tris (ph 9.5) buffer gave the best results with most antibodies. Materials and methods Tissues and reagents Tissues were collected from surgically dissected specimens submitted to the Department of Pathology of Chung-buk National University Hospital. All archival materials were routinely fixed in 10% neutral-buffered formalin, and embedded in paraffin wax. Five micrometre-thick sections were collected onto silane-coated slides (Sigma, St Louis, MO, USA). The following AR buffers were tested (listed in Table 1): 0.05 M glycine buffer (ph 2.0), 0.05 M citrate buffer (ph 6.0), 0.1 M acetate buffer (ph 4.0), 0.1 M phosphate buffer (ph 7.0), 0.01 M HEPES buffer (ph 8.0), 0.05 M Tris

410 Seok H. Kim et al. Table 1. Buffer solutions. Buffer Concentration pka Standard solution (M) ph range Glycine 0.05 2.35 2 3 Acetate 0.10 4.76 4 5 Citrate 0.05 3.14/4.77/6.39 3 6 Phosphate 0.10 7.22 6 8 HEPES 0.05 7.55 7 8 Tris HCl 0.05 8.3 7 10 Borate 0.05 9.23 8 10 Table 2. Antibodies. Antibody Clone name Dilution Test tissue ER 6F11 1 : 50 Breast, infiltrating duct PR 16 1 : 100 Breast, infiltrating duct Ki-67 MM1 1 : 100 Breast, infiltrating duct Cyclin D1 P2D11F11 1 : 100 Parathyroid gland, parathyroid adenoma S-100 S1/61/69 1 : 50 Colon, adeno EMA GP1.4 1 : 100 Breast, infiltrating duct SMA Alpha sm 1 : 100 Colon, rhabdomyosarcoma C-erbB2 10A7 1 : 100 Breast, infiltrating duct P53 DO7 1 : 100 Breast, infiltrating duct Kappa chain Kp-53 1 : 150 Lymph node Lambda chain Hp-6054 1 : 300 Lymph node Vimentin V9 1 : 100 Colon, rhabdomyosarcoma Desmin DE-R-11 1 : 100 Colon, rhabdomyosarcoma CEA 85A12 1 : 100 Colon, rhabdomyosarcoma CD79a 11E3 1 : 200 Lymph node, normal CD3 Ps1 1 : 100 Lymph node, normal Cytokeratin 34 beta E12 1 : 200 Esophagus, squamous cell CK 19 A53-B/A2 1 : 400 Colon, adeno CK 18 DC-10 1 : 800 Colon, adeno CD56 1B6 1 : 100 Pancreas, islet cell tumor (Insulinoma) CD99 DN16 1 : 800 Pancreas, normal Pan cytokeratin Cocktail 1 : 400 Colon, adeno CK 20 Ks20.8 1 : 100 Colon, adeno CK 7 LP5K 1 : 80 Kidney, transitional cell EGFR EGFR.113 1 : 80 Breast, infiltrating duct CD34 QBEND10 1 : 1000 Intestine, GIST Chromogranin LK2H10 1 : 100 Pancreas, normal CD68 KP1 1 : 800 Sarcomatoid CD10 56C6 1 : 100 Burkitts lymphoma AR procedures Microwave-heating Tissue sections on slides were totally immersed in microwave-resistant plastic jars filled with the various AR solutions and heated for 15 min. The heating process was in three phases, namely 2 cycles of 5 min heating and 1 min cooling at room temperature and a final cycle of 5 min heating. The retrieval solutions reached boiling point after 3 min heating in the first cycle and 1 min in the second and third cycles. When necessary, more AR buffer was added after the second cycle to compensate for loss due to boiling over and to prevent the sections drying out. After heating, the slides were left in the plastic jars for further 15 min before washing. A domestic microwave oven (Samsung, Seoul, Korea) with a 700 W output was used at its highest setting. Microwave pressure-cooking The slides were totally immersed in plastic jars filled with the various AR solutions and placed in a hard plastic pressure cooker (Nordic Ware, Minneapolis, MN, USA) containing 300 ml distilled water. The cooker was sealed and heated in the microwave oven for about 20 min until the cooker reached its maximum pressure. It was then heated for another 5 min at maximum pressure. Thereafter, the pressure was reduced and the plastic jar gently cooled in a bath of cold tap water. Immunohistochemical staining Tissue sections were deparaffinized with xylene, hydrated in a descending ethanol series and immersed in 3% H 2 O 2 to destroy endogenous peroxidase activity. AR was carried out as described above. The sections were then incubated with primary antibodies for 60 min, rinsed three times with washing buffer, followed by incubation with biotinylated goat anti-mouse antibodies (DiNonA, Seoul, Korea) for a further 20 min. After a rinse, the sections were incubated with horseradish peroxidase-conjugated streptavidin (DiNonA) for 20 min at room temperature. The slides were washed and the chromogen developed for 5 min with 3,3 -diaminobenzidine solution (DiNonA). They were finally counterstained with Meyer s haematoxylin, dehydrated and mounted in Canada balsam. Distilled water containing 0.1% Tween-20 was used as the rinse solution (Kim et al. 2003). AR solutions buffer (ph 9.5) and 0.05 M borate buffer (ph 8.0). All chemicals except HEPES were purchased from Sigma. HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid) was purchased from Amresco (Solon, Ohio, USA). The immunostaining kit and all antibodies (listed in Table 2) were supplied by DiNona Inc (Seoul, Korea). Except for the Tris and glycine buffers, AR buffers (Table 1) were freshly prepared as described in Guide to Protein Purification (Stoll & Blanchard 1990) without addition of acid or alkali for adjusting the ph, and were used without further dilution. The ph of the Tris and glycine buffers was adjusted by adding 10 N HCl and 2 N NaOH, respectively.

Antigen retrieval buffer systems 411 Evaluation of the results of immunostaining Staining intensity was classified as:, no staining; ±, focal and weakly positive; +, diffuse and weakly positive; ++, diffuse and moderately positive; +++, diffuse and strongly positive and ++++, very strong in all cells. Each section was examined and evaluated separately by three pathologists (SHK, MCK, HGS), and where there was a discrepancy this was discussed until unanimity was achieved. Comparisons of immunostaining intensity were carried out at the same position on each tissue section in order to avoid any variation due to heterogeneity of protein distribution. Results Intensity of immunostaining achieved with different AR solutions The results are summarized in Tables 3 and 4. They reveal that citrate buffer ph 6.0 is not the best AR solution for the majority of antibodies, and the optimal AR buffer depends on the particular antibody. Glycine buffer ph 2.0 is very successful for improving AR efficacy for many antibodies, but causes serious side effects, such as loss of tissue and poor morphological fidelity, with almost all the antibodies tested. Acetate buffer ph 4.0 is no better for AR than distilled water serving as a negative control. Phosphate buffer ph 7.0 is less effective than citrate buffer ph 6.0, Tris buffer ph 9.5 and borate buffer ph 8.0. These results suggest that citrate, HEPES, Tris and borate buffers are all acceptable AR solutions for immunostaining (Figure 1, Table 3). Borate buffer appears to be the most effective for many antibodies. However, it tends to increase the endogenous biotin-like activity of some tissues, and a biotin blocking step prior to incubation with primary antibody is therefore required (Kim et al. 2002). Shi et al. (1995) classified antibodies into three groups, depending on how ph influenced their AR staining pattern. Some antibodies that we tested could be placed in the same groups, as follows: Type A (stable type) shows no significant variation in AR staining patterns as the ph of the AR solution is increased from 2 to 9.5. This was true for antibodies against chromogranin, pancytokeratin and vimentin. Type B antibodies (V-type) show a dramatic decrease in immunoreactivity after AR treatment at neutral ph. There were four antibodies of this type: cyclin D1, kappa chain, lambda chain and cytokeratin (34βE12). Type C antibodies (ascending type), in contrast to type B, fail to give any staining or produce only very weak focal staining after AR treatment at low ph. The intensity of their immunostaining then gradually increases with ph. Ki-67 and c-erbb2 antibodies were in this category. However, it is difficult to evaluate the effect of ph on its own on AR because we used different buffers for each ph, and some of the antibodies we examined did not belong to any of the above three categories. Therefore the chemical composition of the AR buffer has an effect on AR immunoassays as well as its ph. Microwave oven irradiation versus microwave pressure-cooking We employed microwave irradiation and microwave pressure-cooking as representative heat-induced AR methods, and compared the efficacy of the two methods (Table 3). In some cases microwave pressure-cooking was slightly more effective than the microwave oven method in terms of intensity of the resultant AR-induced immunostaining, but we observed no obvious differences in the cytological detail preserved. However, since microwave pressure-cooking led to a severe tissue loss with glycine buffer at ph 2, we used glycine buffer at ph 3 instead. In addition, microwave pressure-cooking appeared to increase endogenous biotin-like activity in tissues such as kidney, liver and salivary gland, especially when borate buffer was used. Discussion In this study, we examined the influence of different AR buffers on the heat-induced immunostaining of 29 antibodies that are frequently used in hospital practice. We tested seven AR buffers whose ph ranged from 2.0 to 9.5, and in most cases AR efficacy was found to be greatly affected by the ph as well as on the chemical composition of the AR buffer. Of these buffers, borate buffer ph 8.0 and Tris buffer ph 9.5 appear to be the most effective in the majority of cases. However, the optimal AR solution differed for different antibodies, and therefore the best AR solution should be sought for each antibody. The optimal AR solutions, based on our results, for each of the antibodies we investigated are given in Table 4. Microwave pressure-cooking resulted in a slightly better AR efficacy than the microwave oven method. It has been shown previously that the ph of the AR solution as well as heating time and temperature are critical factors in heat-induced AR (Evers & Uylings 1994, Shi et al. 1995). Testing a battery of solutions has been recommended as the most practical way for optimizing a protocol (Taylor et al. 1996a,b, Shi et al. 1997). However, it is impractical and unrealistic for each hospital immunohistochemistry laboratory to apply a battery of tests for each of the more than 50 antibodies they commonly use. The test that they proposed employed only one buffer, Tris HCl or citrate, to cover the whole ph range from 1 to 10, and in such a situation the buffer capacity can be limiting. Moreover, since the AR solution can be affected by external factors such as carbon dioxide in the air and temperature, it needs to be prepared freshly and the ph measured each time. The ph of Tris HCl buffer is particularly liable to change on storage at 4 C. Generally, the capacity of a buffer is greatest at its pka and drops off quickly at 1 ph unit on either side of this value (Stoll & Blanchard 1990). In practice, buffers should not be used outside these values. Moreover, the use of stable buffers permits the preparation of large quantities at a time.

412 Seok H. Kim et al. Table 3. Effects of ph and AR buffers on AR employing the microwave and pressure-cooking procedures. Procedure Buffer Antibody ER PR Ki67 Cyclin D1 S-100 EMA SMA C-erbB2 Kappa chain Lambda chain Clone name 6F11 16 MM1 P2D11F11 S1/61/69 GP1.4 Alpha sm 10A7 Kp-53 Hp-6054 Dilution 1 : 50 1 : 100 1 : 100 1 : 100 1 : 50 1 : 100 1 : 100 1 : 100 1 : 150 1 : 300 Microwave Distilled water ± + ++ ± Glycine ph 2 +R ++R ++R ++R +R R ++++R R +++R +++R (B/G+) (B/G+) (B/G+) (B/G+) (B/G+++) Citrate ph 6 ++ +++ ++ +++ +++ +++ + ++ ± Acetate ph 4 ± + ++ R ++ ± Phosphate ph 7 + ++ ++ + ++ ± ± HEPES ph 8 + +++ ++ +++ +++ +++ + ++ + Tris HCl ph 9.5 ++ +++ +++ ++ +++ +++ ++ ++ +++ + Borate ph 8 +++ ++++ +++ +++ +++ +++ +++ +++ +++ ++ Pressure cooking Distilled water ± + + ++ ++ ± ± Glycine ph 3 +R ++R ++R +++R ++R R ++++R R +++R +++R (B/G+) (B/G+++) Citrate ph 6 +++R +++ +++ +++ +++ +++ ++ ++ + Acetate ph 4 + + ++ ++ ± Phosphate ph 7 +++ + ++ ++ ++R ± ± HEPES ph 8 ++ +++R ++ + +++ +++ +++ + +++ + Tris HCl ph 9.5 +++ ++++ +++ +++ +++ ++++ +++ +++ +++ ++ Borate ph 8 +++ ++++ ++++ ++++ +++ ++++ ++++R +++ +++ ++ Vimentin Desmin CEA CD79a P53 CD3 Cytokeratin CK 19 CK 18 V9 DE-R-11 85A12 11E3 DO7 Ps1 34 beta E12 A53-B/A2 DC-10 1 : 100 1 : 100 1 : 100 1 : 200 1 : 100 1 : 100 1 : 200 1 : 400 1 : 800 Microwave Distilled water ++ + ± + + ± ± ± ± Glycine ph 2 +++R ++++R +R ++R ++R ++++R +++R +++R +++R (B/G+) (B/G+) (B/G++) Citrate ph 6 +++ ++ ++ +++ +++ ++ + ++ +++ Acetate ph 4 ++ + ± ++ ++ ± ++ ± Phosphate ph 7 ++ + ± ++ ++ ++ + + + HEPES ph 8 +++ +++ ++ +++ +++ ++ ++ +++ ++++ Tris HCl ph 9.5 +++R +++ ++ +++ +++ ++ +++ +++ ++++ Borate ph 8 +++ ++++ ++ ++++ ++++ +++ + +++ ++++

Antigen retrieval buffer systems 413 Pressure cooking Distilled water ++ + + ± ++ + ++ ± ± Glycine ph 3 ++R ++++R +R +++R +++R ++++R ++++R ++++R ++++R (B/G+) (B/G+) (B/G+) (B/G++) (B/G++) Citrate ph 6 +++ ++ +++ ++ +++ ++ + +++ ++++ Acetate ph 4 ++ ± ± ± ++ ± + ± Phosphate ph 7 +++ ++ ± ++ ++ ++ ++ ++ ++ HEPES ph 8 +++ +++ +++ +++ +++ ++ ++ ++++ ++++ Tris HCl ph 9.5 +++ ++++ +++ +++ +++ +++ +++ ++++ ++++ Borate ph 8 +++ ++++ +++ ++ ++++ ++++ +++ ++++ ++++ CD56 CD99 Pancyto keratin CK 20 CK 7 EGFR CD34 Chromogranin CD68 CD 10 1B6 DN16 cocktail Ks20.8 LP5K EGFR.113 QBEND10 LK2H10 KP1 56C6 1 : 100 1 : 800 1 : 400 1 : 100 1 : 80 1 : 80 1 : 1000 1 : 100 1 : 800 1 : 100 Microwave Distilled water ++ ++ ± ± ± +++ ++ Glycine ph 2 ++R ++R +++R ++R ++R +++R ++R +++R +++R +++R (B/G+) (B/G++) (B/G++) (B/G++) (B/G++) (B/G++) (B/G++) Citrate ph 6.5 ++ +++ +++ + + ++ ++ +++ +++ ++ Acetate ph 4 + ++ ± + +++ ++ Phosphate ph 7 + ++ +++ + ± ++ ++ +++ + ± HEPES ph 8 ++ +++ +++ ++ ++ +++ ++ ++++ +++ ++ Tris HCl ph 9.5 +++ ++++ +++ +++ ++ +++ ++ +++ +++ ++ Borate ph 8 +++ ++++ +++ +++ +++ +++ ++ +++ ++++ ++ Pressure cooking Distilled water + ++ ± R + ± +++ +++ ± Glycine ph 3 ++R ++R ++++R +++R +R +++R ++R ++R +++R ++R (B/G+) (B/G++) (B/G++) (B/G+) (B/G++) (B/G+) (B/G++) (B/G++) Citrate ph 6.5 ++ +++ +++ + + +++ ++ +++ +++ +++ Acetate ph 4 + ± + + +++ + Phosphate ph 7 + ++ ++ + ± ++ ++ +++ + + HEPES ph 8 ++++ +++ +++ +++ +++ +++ ++ ++++ +++ +++ Tris HCl ph 9.5 ++++ ++++ +++ +++ +++ ++++ ++ +++ ++++ +++ Borate ph 8 ++++ ++++ +++ +++ +++ R ++++ ++ +++ ++++ +++ B/G: Background staining, R: Loss of tissue.

414 Seok H. Kim et al. Table 4. Optimal AR buffers for each antibody. Antibody Clone 1st choice 2nd choice CD3 Ps1 Borate buffer ph 8.0 Tris HCl buffer ph 9.5 Cytokeratin 34 beta E12 Tris HCl buffer ph 9.5 Borate buffer ph 8.0 CK19 A53-B/A2 Borate buffer ph 8.0 Tris HCl buffer ph 9.5 CK18 DC-10 Borate buffer ph 8.0 Tris HCl buffer ph 9.5 CD56 1B6 Borate buffer ph 8.0 Tris HCl buffer ph 9.5 CD99 DN16 Tris HCl buffer ph 9.5 Borate buffer ph 8.0 Pancytokeratin Cocktail Borate buffer ph 8.0 Tris HCl buffer ph 9.5 CK20 Ks20.8 Borate buffer ph 8.0 Tris HCl buffer ph 9.5 CK7 LP5K Borate buffer ph 8.0 Tris HCl buffer ph 9.5 EGFR EGFR.113 Tris HCl buffer ph 9.5 Borate buffer ph 8.0 CD34 QBEND10 Tris HCl buffer ph 9.5 Borate buffer ph 8.0 Chromogranin LK2H10 Citrate buffer ph 6.0 HEPES buffer ph 8.0 CD68 Kp1 Borate buffer ph 8.0 Tris HCl buffer ph 9.5 CD10 56C6 Tris HCl buffer ph 9.5 Borate buffer ph 8.0 ER 6F11 Borate buffer ph 8.0 Tris HCl buffer ph 9.5 PR 16 Tris HCl buffer ph 9.5 Borate buffer ph 8.0 Ki67 MM1 Borate buffer ph 8.0 Tris HCl buffer ph 9.5 Cyclin D1 P2D11F11 Borate buffer ph 8.0 Tris HCl buffer ph 9.5 S-100 S1/61/69 Tris HCl buffer ph 9.5 Borate buffer ph 8.0 EMA GP1.4 Borate buffer ph 8.0 HEPES buffer ph 8.0 SMA Alpha sm Tris HCl buffer ph 9.5 Borate buffer ph 8.0 C-erbB2 10A7 Borate buffer ph 8.0 Tris HCl buffer ph 9.5 P53 DO7 Borate buffer ph 8.0 Tris HCl buffer ph 9.5 Kappa chain Kp-53 Borate buffer ph 8.0 Tris HCl buffer ph 9.5 Lambda chain Hp-6054 Tris HCl buffer ph 9.5 Borate buffer ph 8.0 Vimentin V9 Tris HCl buffer ph 9.5 Citrate buffer ph 6.0 Desmin DE-R-11 Borate buffer ph 8.0 Tris HCl buffer ph 9.5 CEA 85A12 Citrate buffer ph 6.0 Tris HCl buffer ph 9.5 CD79a 11E3 Tris HCl buffer ph 9.5 HEPES buffer ph 8.0 We found that citrate buffer ph 6.0, the most popular AR buffer used in hospital practice and research, is not the best choice for most antibodies. According to Shi et al. (1995), the ph of the AR solution is the critical factor, because citrate, phosphate and acetate buffers give similar staining intensities at the same ph. However, in the present study, the chemical composition as well as the ph turned out to be critical. For example, HEPES buffer ph 8.0 and borate buffer ph 8.0 showed different AR efficacies when used with many antibodies. Moreover, citrate buffer ph 6.0 is surprisingly effective by comparison with the effects of ph reported by Shi et al. (1995). According to these workers, AR solutions of ph 6.0 give generally poorer results than solutions of higher ph, irrespective of buffer type. The unexpected efficacy of citrate and borate buffers confirms the idea that buffer composition, as well as ph, is critical. The reason for the enhanced AR under acidic and basic conditions is unknown. However, it is well known that the hydrolysis of amides increases in both acidic and basic solutions. In the case of basic hydrolysis, hot solutions of hydroxides are sufficiently powerful nucleophiles to attack an amide carbonyl group. In acidic hydrolysis, protonation of the carbonyl group by acid renders it sufficiently electrophilic to be attacked by water and give rise to a neutral tetrahedral intermediate. Acidic hydrolysis is much more complex and has a more rate-limiting step than basic hydrolysis, so that a higher acid concentration is required than in basic hydrolysis (Clayden et al. 2001). This is consistent with our finding that a strong acid is required for AR, whereas a relatively weak base was sufficient. However, it does not agree with the suggestion of Morgan et al. (1997) that changes in Ca 2+ levels are responsible for enhancing AR activity in acidic and basic conditions. In support of that view they demonstrated that the incorporation of calcium into various AR solutions inhibited AR only at elevated ph. However, this observation can also be explained by an interaction of calcium as an electrophile with hydroxide ions as nucleophiles, causing a reduction in alkaline hydrolysis unrelated to any alteration of Ca 2+ levels. At acidic ph levels, calcium fails to inhibit retrieval, indicating that the AR-enhancing effect of low ph cannot be due to a calcium exclusion effect. Instead, the fact that calcium-chelating agents such EDTA and EGTA enhance AR suggests that tight complexing of calcium or other divalent metal cations with proteins during formaldehyde fixation is responsible for masking certain antigens (Morgan et al. 1994). In particular, the AR-enhancing activity of citrate buffer may be due to removal of tissue-bound calcium, and this might account for the unexpectedly high AR efficacy of citrate buffer. In our study, pressure-cooking proved to be slightly superior to the microwave oven method. Our results are generally consistent with previous evidence that pressure-cooking is at least as effective as microwave-heating (Norton et al. 1994, Taylor et al. 1996a,b, Pileri et al. 1997). In addition, pressure-cooking permits the handling of greater numbers

Antigen retrieval buffer systems 415 Figure 1. Immunostaining of normal human lymph nodes with anti-cd3 antibody after AR with various AR buffers. A H: microwave oven method. I P: pressure-cooking. B: glycine buffer ph 2. J: glycine buffer ph 3. C, K: acetate buffer ph 4. D, L: citrate buffer ph 6.5. E, M: phosphate buffer ph 7. F, N: HEPES buffer ph 8. G, O: borate buffer ph 8. H, P: Tris-Hcl buffer ph 9.5. A, I: controls (distilled water). Note that Tris buffer ph 9.5 and borate buffer ph 8.0 are superior to citrate buffer ph 6.5.

416 Seok H. Kim et al. of slides and eliminates the problem of hot and cold spots within the microwave oven, obviating the need to use precisely located Coplin jars. In view of these results, pressurecooking seems to be the preferred option in the majority of cases. Acknowledgements We wish to thank Jung Sun Lee, Ji Hye Yun and Young Uk Park for technical assistance during the preparation of this manuscript. This work was partly supported by the 2003 DiNonA Inc. R&D project, Seoul, Korea. References Cattoretti G, Pileri S, Parravicini C, Becker MHG, Poggi S, Bifulco C, Key G, D amanto L, Sabarrini E, Feudale E, Reynolds F, Gerdes J, Rilke F (1993) Antigen unmasking on formalin-fixed paraffinembedded tissue sections. J Pathol 171: 83 98. Clayden J, Greeves N, Warren S, Wothers P (2001) Organic Chemistry. Oxford, United Kingdom: Oxford University Press, pp. P293 P294. Evers P, Uylings HBM (1994) Microwave-stimulated antigen retrieval is ph and temperature dependent. J Histochem Cytochem 42: 1555 1563. Kim SH, Jung KC, Shin YK, Lee KM, Park YS, Choi YL, Oh KI, Kim MK, Chung DH, Song HG, Park SH (2002) The enhanced reactivity of endogenous biotin-like molecules by antigen retrieval procedures and signal amplification with tyramine. Histochem J 34: 97 103. Kim SH, Shin YK, Lee KM, Lee JS, Yun JH, Lee SH (2003) An improved protocol of biotinylated tyramine-based immunohistochemistry minimizing nonspecific background staining. J Histochem Cytochem 51: 129 132. Morgan JM, Navabi H, Schmid KW, Jasani B (1994) Possible role of tissue-bound calcium ions in citrate mediated high temperature antigen retrieval. J Pathol 174: 301 307. Morgan JM, Navabi H, Jasani B (1997) Role of calcium chelation in high-temperature antigen retrieval at different ph values. J Pathol 182: 233 237. Norton AJ, Jordan S, Yeomans P (1994) Brief, high-temperature heat denaturation (pressure cooking): A simple and effective method of antigen retrieval for routinely processed tissues. J Pathol 173: 371 379. Pileri SA, Roncador G, Ceccarelli C, Piccioli M, Briskomatis A, Sabattini E, Ascani S, Santini D, Piccaluga PP, Leone O, Damiani S, Ercolessi C, Sanri F, Pieri F, Leoncini L, Falini B (1997) Antigen retrieval techniques in immunohistochemistry: comparison of different methods. J Pathol 183: 116 123. Shi SR, Key ME, Kalra KL (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: An enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39: 741 748. Shi SR, Imam SA, Young L, Cote RJ, Taylor CR (1995) Antigen retrieval immunohistochemistry under the influence of ph using monoclonal antibodies. J Histochem Cytochem 43: 193 201. Shi SR, Cote RJ, Taylor CR (1997) Antigen retrieval immunohistochemistry: Past, present, and future. J Histochem Cytochem 45: 327 343. Stoll VS, Blanchard JS (1990) Buffers: Principles and practice. In: Deutscher MP, eds. Guide to Protein Purification. Vol. 182. Methods in Enzymology, 1st edn. San Diego: Academic press, pp. 24 38. Taylor CR, Shi SR, Cote RJ (1996a) Antigen retrieval for immunohistochemistry: Status and need for greater standardization. Appl Immunohistochem 4: 144 166. Taylor CR, Shi SR, Chen C, Young L, Cote RJ (1996b) Comparative study of antigen retrieval heating methods: Microwave, microwave and pressure cooker, autoclave, and steamer. Biotechnic Histochem 71: 263 270.