A Comparison of Methods for Heat-Mediated Antigen Retrieval for Immunoelectron Microscopy: Demonstration of Cytokeratin No. 18 in Normal and Neoplastic Hepatocytes

Postembedding antigen retrieval is a veil established technique for immnoelectron microscopy; however, many antigens cannot be detected without additional unmasking procedures. This study was undertaken to determine whether microwave oven heating, autoclaving, and pressurized boiling, which are well recognized methods of antigen retrieval for light microscopy, and simple boiling can also be used in electron microscopy. We investigated neoplastic and normal hepatocytes using a commercially available mouse monoclonal antibody against cytokeratin NO. 18 (CK18). The tissue was fixed in paraformaldehyde / gintaraldehyde and embedded in Lowicryl K4M at -40 C. Ultrathin sections in various buffers were exposed to heat using one of four methods or to pronase at 37 C before incubation with the primary antibody. The secondary antibody was gold-labeled goat anti-mouse antibody. Sections that were not heat-treated remained unlabeled, but heat-treated sections showed immu-noreactivity located mainly at the cytoplasmic periphery. Some of the gold particles lay in direct or loose association with intermediate filaments, some were seen in the area of desmosomes, and some did not appear related to any structures. No difference in immunostainlng was found among the four methods of heat treatment. The citrate buffer, pH 6.0, and 10 mM EDTA, pH 8.0, generated the best labeling results.     

Antigen retrieval trial for post-embedding immunoelectron microscopy by heating with several unmasking solutions.

A novel antigen retrieval procedure was carried out in the post-embedding immunogold electron microscopy method to improve the stainability of the samples. This was done by weakly fixing cultured Helicobacter pylori (ATCC43504) and embedding in Lowicryl K4M. Before staining with the anti-H. pylori antibody, the ultrathin sections were mounted on a nickel grid and heated at 121C for 15 min, 99C for 40 min, and 65C for 24 hr in distilled water, 0.1 M phosphate buffer (pH 7.4), 0.01 M EDTA (pH 7.2), 0.05 M Tris buffer (pH 10.0), 0.8 M urea (pH 7.2), 0.01 M citric acid (pH 6.0), or a commercially available target unmasking fluid (S1699; pH 6.0). Antigen retrieval in the Tris buffer solution generally showed better stainability than the classical post-embedding method without any antigen retrieval. At 65C for 24 hr, better stainability of the ultrasections was observed for each of the solutions used except for the phosphate buffer compared to the control. We suggest that the antigen retrieval method should be applied for routine use even by in post-embedding immunogold electron microscopy.     

DNA extraction from archival formalin-fixed,paraffin-embedded tissues: heat-induced retrieval in alkaline solution

Based on the antigen retrieval principle, our previous study has demonstrated that heating archival formalin-fixed, paraffin-embedded (FFPE) tissues at a higher temperature and at higher pH value of the retrieval solution may achieve higher efficiency of extracted DNA, when compared to the traditional enzyme digestion method. Along this line of heat-induced retrieval, this further study is focused on development of a simpler and more effective heat-induced DNA retrieval technique by testing various retrieval solutions. Three major experiments using a high temperature heating method to extract DNA from FFPE human lymphoid and other tissue sections were performed to compare: (1) different concentrations of alkaline solution (NaOH or KOH, pH 11.5–12) versus Britton and Robinson type of buffer solution (BR buffer) of pH 12 that was the only retrieval solution tested in our previous study; (2) several chemical solutions (SDS, Tween 20, and GITC of various concentrations) versus BR buffer or alkaline solution; and (3) alkaline solution mixed with chemicals versus BR buffer or single alkaline solution. Efficiency of DNA extraction was evaluated by measuring yields using spectrophotometry, electrophoretic pattern, semiquantitation of tissue dissolution, PCR amplification, and kinetic thermocycling-PCR methods. Results showed that boiling tissue sections in 0.1 M NaOH or KOH or its complex retrieval solutions produced higher yields and better quality of DNA compared to BR buffer or chemical solutions alone. The conclusion was that boiling FFPE tissue sections in 0.1 M alkaline solution is a simpler and more effective heat-induced retrieval protocol for DNA extraction. Combination with some chemicals (detergents) may further significantly improve efficiency of the heat-induced retrieval technique.     

系统解剖学立体化双语教学资源建设的探讨

系统解剖学是医学生最为重要的专业基础课程之一。近年来,我们一直坚持双语教学,2008年我校系统解剖学获得首批国家双语教学示范课程。为建设好双语教学示范课程,我们积极开展解剖学双语教学探索和实践,着重于该门课程的立体化双语教学资源的建设,以纸质教材为主体,利用不同教学媒体的优点来呈现解剖学不同的教学内容,形成一个立体化的双语教学资源,并应用于教学实践活动中,达到提高学生专业英语理解能力,灵活运用外语思维解决医学问题能力的目标。     

一种改良的BrdU免疫组织化学染色抗原热修复技术

目的：探索一种简单有效的行BrdU免疫组织化学染色抗原热修复的方法。方法：本实验探索了三种抗原热修复方法：1）漂片煮沸法,2）贴片煮沸法,3）贴片改良法。将贴有冰冻组织切片的玻片置于恒温封闭体系中进行热修复。之后行BrdU免疫组化染色,栗集图像对这三种热修复方法进行比较。针对贴片改良法行BrdU与NeuN、P。CERB和DCX的荧光双标,采集图像以观察该方法在免疫荧光甄标实验中应用的可行性。结果：1）漂片煮沸法脑组织切片在经过热修复处理后,切片卷起皱缩,不能进行后续实验步骤;2）贴片煮沸法可见少量BrdU阳性细胞,但背景深,且少数脑片起泡,假阳性现象严重;3）贴片改良法B-U免疫组化及与NeuN、P-CERB和DCX的免疫荧光双标染色背景浅,阳性细胞明显。结论：采用改良的热修复法能充分暴露BrdU抗原,DAB显色及免疫荧光染色结果理想。此方法为一种较好的行BrdU免疫组织化学染色抗原热修复方法。     

Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.

We describe a new approach for retrieval of antigens from formalin-fixed, paraffin-embedded tissues and their subsequent staining by immunohistochemical techniques. This method of antigen retrieval is based on microwave heating of tissue sections attached to microscope slides to temperatures up to 100 degrees C in the presence of metal solutions. Among 52 monoclonal and polyclonal antibodies tested by this method, 39 antibodies demonstrated a significant increase in immunostaining, nine antibodies showed no change, and four antibodies showed reduced immunostaining. In particular, excellent immunostaining results were obtained with a monoclonal antibody to vimentin as well as several different keratin antibodies on routine formalin-fixed tissue sections after pre-treatment of the slides with this method. These results showed that after antigen retrieval: (a) enzyme predigestion of tissues could be omitted; (b) incubation times of primary antibodies could be significantly reduced, or dilutions of primary antibodies could be increased; (c) adequate staining could be achieved in long-term formalin-fixed tissues that failed to stain by conventional methods; and (d) certain antibodies which were typically unreactive with formalin-fixed tissues gave excellent staining.     

An antigen retrieval method using an alkaline solution allows immunoelectron microscopic identification of secretory granules in conventional epoxy-embedded tissue sections.

Immunoelectron microscopy using chromogranin A-specific antibodies has been proposed as an efficient technique for identification of secretory granules (SGs) in tumor cells with evidence of apparent neuroendocrine differentiation. Using an antigen retrieval (AR) method, we succeeded in immunolabeling SGs with antibodies in ultrathin sections of routinely processed epoxy-embedded blocks of tissue. Samples of an insulinoma were fixed in 2% glutaraldehyde, postfixed in 1% OsO(4), and embedded in epoxy resin. Ultrathin sections were immunostained with chromogranin A-specific antibodies and gold-conjugated second antibodies. There was no significant labeling in the absence of AR. Neither etching with sodium metaperiodate nor microwave irradiation of ultrathin sections in citrate buffer (pH 6.0) or in EDTA buffer (pH 8.0) was effective in improving the efficiency of immunolabeling. However, ultrathin epoxy-embedded sections that were microwaved in alkaline solution (pH 10) were adequately labeled (5.2 +/- 0.34 particles per SG). Moreover, considerably improved efficiency of immunostaining was achieved by microwaving sections in alkaline solution (pH 10) with subsequent immunostaining at 60C (12.2 +/- 0.51 particles per SG). This method can also be applied to epoxy-embedded sections obtained from formalin-fixed, paraffin-embedded blocks of tissue and was even valid for an old epoxy-embedded block of tissue prepared 15 years previously.     

Mechanisms of heat-induced antigen retrieval: does pH or ionic strength of the solution play a role for refolding antigens?

We investigated the effects of pH and ionic strength of solutions used for antigen retrieval to elucidate the mechanism of heat-induced antigen retrieval (HIAR) in immunohistochemistry. The immunostaining intensity of nuclear, cytoplasmic, cell membrane, and extracellular matrix antigens with 17 different antibodies was evaluated in formaldehyde-fixed and paraffin-embedded mouse and human tissues. Deparaffinized sections were autoclaved for 10 min in buffers with different pH values ranging from 3.0 to 10.5. To test the influence of ionic strength on immunoreactions, the sections were autoclaved for 10 min in 20 mM Tris-HCl buffers (TB) at pH 9.0 and 10.5 with or without 25, 50, and 100 mM NaCl. There were two immunostaining patterns for pH dependency of HIAR. First, the majority of antibodies recovered their antigenicity when heated in the buffers with both acidic pH (pH 3.0) and basic pH (pH 9.0 and 10.5). Second, some antibodies showed strong immunostaining only at basic pH values (pH 9.0 and 10.5). When the sections were autoclaved in TB at pH 9.0, immunostaining of all eight antibodies examined decreased as the NaCl concentration increased. On the other hand, when the sections were treated with TB at pH 10.5, all antibodies yielded stronger reactions in the buffer containing NaCl than in the buffer without NaCl; five antibodies exhibited the strongest immunoreaction at concentrations from 25 to 50 mM. These results suggest that the extended polypeptides by heating are charged negatively or positively at basic or acidic pH, and that an electrostatic repulsion force acts to prevent random entangling of polypeptides caused by hydrophobic attractive force and to expose antigenic determinants, during cooling process of HIAR solution.     

Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.     

A Single Simple Procedure for Dewaxing,Hydration and Heat-Induced Epitope Retrieval (HIER) for Immunohistochemistry in Formalin-Fixed Paraffin-Embedded Tissue

Heat-induced epitope retrieval (HIER) is widely used for immunohistochemistry on formalin-fixed paraffin-embedded tissue and includes temperatures well above the melting point of paraffin. We therefore tested whether traditional xylene-based removal of paraffin is required on sections from paraffin-embedded tissue, when HIER is performed by vigorous boiling in 10 mM Tris/0.5 mM EGTA-buffer (pH=9). Immunohistochemical results using HIER with or without prior dewaxing in xylene were evaluated using 7 primary antibodies targeting proteins located in the cytosol, intracellular vesicles and plasma membrane. No effect of omitting prior dewaxing was observed on staining pattern. Semiquantitative analysis did not show HIER to influence the intensity of labelling consistently. Consequently, quantification of immune labelling intensity using fluorescent secondary antibodies was performed at 5 dilutions of primary antibody with and without prior dewaxing in xylene. No effect of omitting prior dewaxing on signal intensity was detectable indicating similar immunoreactivity in dewaxed and non-dewaxed sections. The intensity of staining the nucleus with the DNA-stain ToPro3 was similarly unaffected by omission of dewaxing in xylene.In conclusion, the HIER procedure described and tested can be used as a single procedure enabling dewaxing, hydration and epitope retrieval for immunohistochemistry in formalin-fixed paraffin-embedded tissue.Key words: Immunolabelling, antibodies, histology, protein localization, HIER     

Reversing the effects of formalin fixation with citraconic anhydride and heat: a universal antigen retrieval method.

Formalin is a commonly used fixative for tissue preservation in pathology laboratories. A major adverse effect of this fixative is the concealing of tissue antigens by protein cross-linking. To achieve a universal antigen retrieval method for immunohistochemistry under a constant condition, we developed a new method in which the effects of formalin fixation were reversed with citraconic anhydride (a reversible protein cross-linking agent) plus heating. Formalin-fixed, paraffin-embedded tissues from various organs were examined for immunohistochemical localization of a wide variety of antigens. Deparaffinized tissue sections were placed in an electric kitchen pot containing 0.05% citraconic anhydride solution, pH 7.4, and the pot was set at "keep warm" temperature mode of 98C for 45 min. This mode allowed heating the sections at a constant temperature. The sections were then washed in buffer solution and immunostained using a labeled streptavidin-biotin method using an automated stainer. In general, formalin-fixed tissues demonstrated specific immunostainings comparable to that in fresh frozen tissues and significantly more enhanced than after conventional antigen retrieval methods. In particular, even difficult-to-detect antigens such as CD4, cyclin D1, granzyme beta, bcl-6, CD25, and lambda chain revealed distinct immunostainings. Different classes of antigens such as cellular markers and receptors, as well as cytoplasmic and nuclear proteins, consistently produced enhanced reactions. This method provides efficient antigen retrieval for successful immunostaining of a wide variety of antigens under an optimized condition. It also allows standardization of immunohistochemistry for formalin-fixed tissues in pathology laboratories, eliminating inter-laboratory discrepancies in results for accurate clinical and research studies.     

Evaluation of immunohistochemical staining of human duodenal endocrine cells after microwave antigen retrieval

Summary The effect of microwave antigen retrieval on the immunostaining of human duodenal endocrine cells in formaldehyde-fixed, paraffin-embedded material was investigated. The sections were immunostained by the avidin-biot in complex (ABC) and immunogold-silver autometallography (IGSS) methods with and without prior microwave treatment. Dilutions of up to 1:30 000 of the following antisera/antibodies were used: anti-chromogranin A, anti-chromogranin AB, anti-secretin, anti-gastrin, anti-gastric inhibitory polypeptide, anti-somatostatin and anti-serotonin. The detection threshold for all the antibodies was lower after antigen retrieval, and the primary antibody could be used in higher dilutions. The dilutions varied for different antibodies and were between two and ten times the optimal dilution without antigen retrieval. At extremely high dilutions of, or without, the primary antibody, non-specific staining of some lymphocytes and the mucus of some goblet cells was observed when the avidin method was applied, but not with the immunogold technique. This phenomenon was not observed when optimal dilution or a lower dilution was used. This seems to have been caused by the binding of the avidin-biotin complex to epitopes in these structures unmasked by microwave treatment when competition with specific binding sites was absent. This revised version was published online in November 2006 with corrections to the Cover Date.     

Antigen retrieval by heating en bloc for pre-fixed frozen material.

Antigen retrieval (AR) is frequently required for successful immunohistochemistry (IHC) in archival formalin-fixed, paraffin-embedded tissue sections. Although AR by heating is most generally used, the majority of existing methods are useful only for paraffin-embedded sections. This article describes a simple alternative method for AR that can be used for aldehyde-fixed frozen sections. After fixation in paraformaldehyde, tissue blocks were heated in retrieval solutions and then frozen with dry ice. The optimal temperatures for heating were 90C and above, and the optimal retrieval solutions were distilled water and 10 mM sodium citrate, pH 6.0. Sections were cut with a cryostat and mounted on poly-l-lysine-coated glass slides. After the sections dried, routine IHC was performed. Alternatively, free-floating sections were used. This method not only greatly enhanced the immunoreactivity for a wide range of antigens, especially for nuclear proteins, but also effectively lowered the background staining in some cases. I examined the staining of 14 antibodies using sections of mouse brain and rat testis. The heating process was essential for five antibodies, improved immunoreactivity for seven antibodies, and provided no change for two antibodies.     

Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS)

A simple method for antigen retrieval in tissue sections and cell cultures is described. Because many antibodies recognize denatured proteins on western blots, but are poorly reactive by immunocytochemistry, the effect of applying sodium dodecyl sulfate (SDS) to cryostat sections of tissues and to cell cultures prior to immunostaining was examined. In many cases, a 5-min pretreatment with 1% SDS produced a dramatic increase in staining intensity by indirect immunofluorescence. Among the antibodies tested that showed a positive effect of SDS were an anti-Na/K-ATPase monoclonal antibody, an anti-AE1/2 anion exchanger polyclonal antipeptide antibody, a monoclonal anti-caveolin antibody, and an anti-rab4 monoclonal antibody. In other cases, including antibodies against gp330, aquaporin 1, and aquaporin 2, no effect of SDS was detected. The results show that SDS treatment can be used as a simple method of antigen retrieval in cryostat sections and on cultured cells. In some cases, antigens were not detectable without pretreatment with SDS.     

Development of an optimal antigen retrieval protocol for immunohistochemistry of retinoblastoma protein (pRB) in formalin fixed, paraffin sections based on comparison of different methods

A novel protocol for antigen retrieval (AR) for immunohistochemistry (IHC) of retinoblastoma protein (pRB) in formalin fixed, paraffin embedded (FFPE) tissue sections was developed using 0.05% citraconic anhydride as the AR solution for heat treatment based on comparison of different methods. This new protocol has advantages including superior morphological preservation, greater reproducibility, and more intense staining after retrieval. Our study demonstrates the importance of comparing various AR protocols to obtain maximal IHC for standardization and for quantitative IHC.     

Development of an optimal antigen retrieval protocol for immunohistochemistry of retinoblastoma protein (pRB) in formalin fixed,paraffin sections based on comparison of different methods

A novel protocol for antigen retrieval (AR) for immunohistochemistry (IHC) of retinoblastoma protein (pRB) in formalin fixed, paraffin embedded (FFPE) tissue sections was developed using 0.05% citraconic anhydride as the AR solution for heat treatment based on comparison of different methods. This new protocol has advantages including superior morphological preservation, greater reproducibility, and more intense staining after retrieval. Our study demonstrates the importance of comparing various AR protocols to obtain maximal IHC for standardization and for quantitative IHC.     

Antigen Retrieval Using pH 3.5 Glycine-HCI Buffer or Urea Solution for Immunohistochemical Localization of Ki-67

A new antibody (MIB-1) has been described, permitting the demonstration of Ki-67 proliferation antigen in paraffin sections. However, satisfactory results were obtained only after subjecting tissue sections to microwave based antigen retrieval in citrate buffer solution. Other buffer solutions produce equivalent or better results and also permit use of the original Ki-67 antibody, which hitherto has been considered ineffective for paraffin sections.     

Use of pH 9.5 Tris-HCI Buffer Containing 5% Urea for Antigen Retrieval Immunohistochemistry

Successful antigen retrieval (AR) immunohistochemistry is dependent on the temperature, heating time, and pH value of the AR solutions. There is no single standardized AR solution, however, that is suitable for all antibodies “routinely” used in surgical pathology for immunostaining archival tissue sections. We tested a variety of AR solutions varying in pH value, chemical composition, and molarity. Based upon preliminary results, we compared three AR solutions: 0.1 M Tris-HCI buffer, pH 9.5, containing 5% urea, 0.1 M Tris-HCI buffer pH 9.5 without urea, and citrate buffer, pH 6.0. Each AR solution was tested with a panel of 34 antibodies using microwave heating for antigen retrieval. The heating conditions were standardized at 10 min and an automated stainer was used to standardize the immunostaining method. The Tris-HC1 containing urea was superior to pH 6.0 citrate buffer for 22 antibodies. In 12 cases, Tris-HC1 with urea was also superior to Tris-HC1 alone. In 12 cases, the intensity was similar for all three retrieval solutions. The staining obtained with Tris-HC1 with urea was equal to or better than with pH 6.0 citrate buffer in all cases. The Tris-HC1 with urea solution is satisfactory for AR of most antibodies employed in routine surgical pathology.