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Clive R Taylor - One of the best experts on this subject based on the ideXlab platform.
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comparative study of antigen retrieval heating methods microwave microwave and pressure cooker autoclave and steamer
Biotechnic & Histochemistry, 1996Co-Authors: Clive R Taylor, Chen Chen, Lillian Young, Christina Yang, Richard J CoteAbstract:We present a study comparing the most popular beating methods currently used for antigen retrieval (AR) Immunostaining: the microwave oven, microwave with pressure cooker, autoclave, and steamer heating. A panel of 21 antibodies was tested on formalin fixed, paraffin embedded sections using these heating methods and Tris-HC1 buffer, pH 9.5, plus 5% urea as the AR solution. Three observers independently evaluated the intensity of AR Immunostaining. All heating methods yielded good results for AR Immunostaining. There were only minor differences among the heating methods for AR when the optimal concentration of primary antibody for normal Immunostaining was used; however, background staining may occasionally be troublesome if antibodies are not retitrated and diluted further for use on tissues after AR. Significant differences were observed only after further dilution of the primary antibodies; the microwave pressure cooker, extended microwave heating (5 min × 4) and autoclave heating then showed a similar ...
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Comparative Study of Antigen Retrieval Heating Methods: Microwave, Microwave and Pressure Cooker, Autoclave, and Steamer
Biotechnic & histochemistry : official publication of the Biological Stain Commission, 1996Co-Authors: Clive R Taylor, Chen Chen, Christina Yang, Shan Rong Shi, Lillian L. Young, Richard J CoteAbstract:We present a study comparing the most popular heating methods currently used for antigen retrieval (AR) Immunostaining: the microwave oven, microwave with pressure cooker, autoclave, and steamer heating. A panel of 21 antibodies was tested on formalin fixed, paraffin embedded sections using these heating methods and Tris-HC1 buffer, pH 9.5, plus 5% urea as the AR solution. Three observers independently evaluated the intensity of AR Immunostaining. All heating methods yielded good results for AR Immunostaining. There were only minor differences among the heating methods for AR when the optimal concentration of primary antibody for normal Immunostaining was used; however, background staining may occasionally be troublesome if antibodies are not retitrated and diluted further for use on tissues after AR. Significant differences were observed only after further dilution of the primary antibodies: the microwave pressure cooker, extended microwave heating (5 min x 4) and autoclave heating then showed a similar intensity of staining that was stronger than results obtained with the steamer (20 min) or regular microwave heating (5 min x 2). Extension of the steamer heating time, however, yielded equivalent results. This study indicates that different heating methods can yield similar intensities of AR Immunostaining if the heating times are adjusted appropriately. It is noteworthy that, in general, the adjusted conditions for maximal retrieval differ from those most widely cited in the literature, or recommended by manufacturers. That several heating devices may provide similar results permits the use of different AR heating methods according to the equipment available. This study also is an early step in standardizing the AR Immunostaining protocol by providing uniform conditions for "maximal retrieval" as a common end point for all laboratories.
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antigen retrieval immunohistochemistry under the influence of ph using monoclonal antibodies
Journal of Histochemistry and Cytochemistry, 1995Co-Authors: Shan Rong Shi, Richard J Cote, Lillian Young, S A Imam, Clive R TaylorAbstract:Antigen retrieval (AR) incorporating high-temperature microwave (MW) heating of tissue sections before Immunostaining is a revolutionary technique that can unmask the antigens in formalin-fixed tissue sections, thus making them available for immunohistochemical staining. Although high temperature is believed to be the primary mechanism in retrieval of antigens, a variety of chemical solutions have been tested to define an optimal AR solution. We tested the hypothesis that pH of the AR solution may influence the quality of Immunostaining by using seven different AR buffer solutions at a series of different pH values ranging from 1 to 10. We evaluated the staining of monoclonal antibodies to cytoplasmic antigens (AE1, HMB45, NSE), nuclear antigens (MIB-1, PCNA, ER), and cell surface antigens (MT1, L26, EMA) on routinely formalin-fixed, paraffin-embedded sections under different pH conditions with MW heating for 10 min. The intensity of Immunostaining was graded in a blinded fashion. The pH value of the AR buffer solution was carefully measured before, immediately after, and 15 min after the AR procedure. The influence of pH on AR immunohistochemical staining can be summarized into three patterns. Some antigens (L26, PCNA, AE1, EMA, and NSE) showed excellent retrieval throughout the pH range. Other antigens (MIB1 and ER) showed strong intensity of immunohistochemical staining at very low pH and at neutral to high pH, but a dramatic decrease in the intensity of the AR Immunostaining at moderately acidic pH (pH 3-6). Still others (MT1 and HMB45) showed increasing intensity of the AR Immunostaining with increasing pH, but only weak Immunostaining at low pH. Among the seven buffer solutions at any given pH value, the intensity of AR Immunostaining was very similar. However, Tris-HCl buffer tended to produce better results at higher pH, compared with other buffers. Although high-temperature heating is believed to be the most important factor for the AR technique, the pH value of the AR solution is an important co-factor for some antigens. Optimization of the AR system should therefore include optimization of the pH of the AR solution. Our results indicate that AR Immunostaining of Tris-HCl or sodium acetate buffer at pH 8-9 may be suitable for most antigens, although certain nuclear antigens show optimal staining at low pH.
Richard J Cote - One of the best experts on this subject based on the ideXlab platform.
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comparative study of antigen retrieval heating methods microwave microwave and pressure cooker autoclave and steamer
Biotechnic & Histochemistry, 1996Co-Authors: Clive R Taylor, Chen Chen, Lillian Young, Christina Yang, Richard J CoteAbstract:We present a study comparing the most popular beating methods currently used for antigen retrieval (AR) Immunostaining: the microwave oven, microwave with pressure cooker, autoclave, and steamer heating. A panel of 21 antibodies was tested on formalin fixed, paraffin embedded sections using these heating methods and Tris-HC1 buffer, pH 9.5, plus 5% urea as the AR solution. Three observers independently evaluated the intensity of AR Immunostaining. All heating methods yielded good results for AR Immunostaining. There were only minor differences among the heating methods for AR when the optimal concentration of primary antibody for normal Immunostaining was used; however, background staining may occasionally be troublesome if antibodies are not retitrated and diluted further for use on tissues after AR. Significant differences were observed only after further dilution of the primary antibodies; the microwave pressure cooker, extended microwave heating (5 min × 4) and autoclave heating then showed a similar ...
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Comparative Study of Antigen Retrieval Heating Methods: Microwave, Microwave and Pressure Cooker, Autoclave, and Steamer
Biotechnic & histochemistry : official publication of the Biological Stain Commission, 1996Co-Authors: Clive R Taylor, Chen Chen, Christina Yang, Shan Rong Shi, Lillian L. Young, Richard J CoteAbstract:We present a study comparing the most popular heating methods currently used for antigen retrieval (AR) Immunostaining: the microwave oven, microwave with pressure cooker, autoclave, and steamer heating. A panel of 21 antibodies was tested on formalin fixed, paraffin embedded sections using these heating methods and Tris-HC1 buffer, pH 9.5, plus 5% urea as the AR solution. Three observers independently evaluated the intensity of AR Immunostaining. All heating methods yielded good results for AR Immunostaining. There were only minor differences among the heating methods for AR when the optimal concentration of primary antibody for normal Immunostaining was used; however, background staining may occasionally be troublesome if antibodies are not retitrated and diluted further for use on tissues after AR. Significant differences were observed only after further dilution of the primary antibodies: the microwave pressure cooker, extended microwave heating (5 min x 4) and autoclave heating then showed a similar intensity of staining that was stronger than results obtained with the steamer (20 min) or regular microwave heating (5 min x 2). Extension of the steamer heating time, however, yielded equivalent results. This study indicates that different heating methods can yield similar intensities of AR Immunostaining if the heating times are adjusted appropriately. It is noteworthy that, in general, the adjusted conditions for maximal retrieval differ from those most widely cited in the literature, or recommended by manufacturers. That several heating devices may provide similar results permits the use of different AR heating methods according to the equipment available. This study also is an early step in standardizing the AR Immunostaining protocol by providing uniform conditions for "maximal retrieval" as a common end point for all laboratories.
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antigen retrieval immunohistochemistry under the influence of ph using monoclonal antibodies
Journal of Histochemistry and Cytochemistry, 1995Co-Authors: Shan Rong Shi, Richard J Cote, Lillian Young, S A Imam, Clive R TaylorAbstract:Antigen retrieval (AR) incorporating high-temperature microwave (MW) heating of tissue sections before Immunostaining is a revolutionary technique that can unmask the antigens in formalin-fixed tissue sections, thus making them available for immunohistochemical staining. Although high temperature is believed to be the primary mechanism in retrieval of antigens, a variety of chemical solutions have been tested to define an optimal AR solution. We tested the hypothesis that pH of the AR solution may influence the quality of Immunostaining by using seven different AR buffer solutions at a series of different pH values ranging from 1 to 10. We evaluated the staining of monoclonal antibodies to cytoplasmic antigens (AE1, HMB45, NSE), nuclear antigens (MIB-1, PCNA, ER), and cell surface antigens (MT1, L26, EMA) on routinely formalin-fixed, paraffin-embedded sections under different pH conditions with MW heating for 10 min. The intensity of Immunostaining was graded in a blinded fashion. The pH value of the AR buffer solution was carefully measured before, immediately after, and 15 min after the AR procedure. The influence of pH on AR immunohistochemical staining can be summarized into three patterns. Some antigens (L26, PCNA, AE1, EMA, and NSE) showed excellent retrieval throughout the pH range. Other antigens (MIB1 and ER) showed strong intensity of immunohistochemical staining at very low pH and at neutral to high pH, but a dramatic decrease in the intensity of the AR Immunostaining at moderately acidic pH (pH 3-6). Still others (MT1 and HMB45) showed increasing intensity of the AR Immunostaining with increasing pH, but only weak Immunostaining at low pH. Among the seven buffer solutions at any given pH value, the intensity of AR Immunostaining was very similar. However, Tris-HCl buffer tended to produce better results at higher pH, compared with other buffers. Although high-temperature heating is believed to be the most important factor for the AR technique, the pH value of the AR solution is an important co-factor for some antigens. Optimization of the AR system should therefore include optimization of the pH of the AR solution. Our results indicate that AR Immunostaining of Tris-HCl or sodium acetate buffer at pH 8-9 may be suitable for most antigens, although certain nuclear antigens show optimal staining at low pH.
Dominique Wendum - One of the best experts on this subject based on the ideXlab platform.
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reply to the sannier et al letter entitled mdr3 Immunostaining on frozen liver biopsy is not a sensitive diagnosis tool for the detection of heterozygous mdr3 abcb4 gene mutation
Virchows Archiv, 2012Co-Authors: Dominique Wendum, Veronique Barbu, Olivier Rosmorduc, Lionel Arrive, Jeanfrancois Flejou, Raoul PouponAbstract:Sir or Madam, We read with great interest the letter by Sannier et al. The data presented are consistent with our publication [1]. The authors studied a new series of adult patients with unexplained cholestasis and heterozygous MDR3/ABCB4 mutations. In five cases, a MDR3 Immunostaining was performed on liver frozen sections and showed an intense canalicular staining similar to a normal staining. These results are in contradiction with their initial report that showed a faint MDR3 Immunostaining in four patients with heterozygous MDR3/ABCB4 mutations [2]. This discrepancy may be explained by technical hazards or by the type of MDR3/ABCB4 mutations (indeed, the mutations were different in the two series). It could be interesting to perform MDR3 Immunostainings on the formalin-fixed paraffin-embedded liver tissues in these two series that would enable a comparison with our published data which were obtained with this technique. To conclude, it seems that MDR3/ABCB4 heterozygous mutation diagnosis requires gene sequencing.
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aspects of liver pathology in adult patients with mdr3 abcb4 gene mutations
Virchows Archiv, 2012Co-Authors: Dominique Wendum, Veronique Barbu, Olivier Rosmorduc, Lionel Arrive, Jeanfrancois Flejou, Raoul PouponAbstract:The aims of this study were to describe the histological liver lesions in adult patients with MDR3/ ABCB4 mutation and to study the usefulness of MDR3 Immunostaining as a diagnostic tool. All adult patients from our institution with an MDR3/ABCB4 mutation and a liver histology were included (n = 13). Eleven patients had a single heterozygous gene mutation and two patients had two heterozygous mutations. Two patients had no liver lesions. Eight patients had a mild ductular reaction and portal fibrosis. One patient had a few fibrous septa and two patients had biliary cirrhosis. In three cases intraductal lipid crystals were identified. Two patients had biliary fibroobliterative lesions with no sclerosing cholangitis on cholangiography. Biliary dysplasia was identified in hepatectomy specimens from two patients, one of whom developed an intrahepatic cholangiocarcinoma. One patient with biliary cirrhosis developed a hepatocellular carcinoma. MDR3 Immunostainings performed on formalin-fixed paraffin-embedded sections showed a strong canalicular staining in all patients except in one. To conclude, the predominant histological features were ductular reaction with no or mild fibrosis without cholangitis. Liver lesions previously unreported in association with MDR3/ABCB4 gene mutations (biliary dysplasia, cholangiocarcinoma, small duct sclerosing cholangitis) were also found. Lipid crystals in bile ducts may be suggestive of MDR3/ABCB4 mutation. MDR3 Immunostaining on formalin-fixed paraffin-embedded sections does not seem to be sensitive for the diagnosis of heterozygous MDR3/ABCB4 mutations.
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regressive liver adenomatosis following androgenic progestin therapy withdrawal a case report with a 10 year follow up and a molecular analysis
European Journal of Endocrinology, 2007Co-Authors: Magali Svrcek, Lionel Arrive, Jeanfrancois Flejou, Raoul Poupon, Emmanuelle Jeannot, Gaelle Fromont, Jessica Zucmanrossi, Philippe Bouchard, Dominique WendumAbstract:Objective: The relationship between sex hormones and hepatocellular adenoma development is well established. On the contrary, their contribution to liver adenomatosis (LA) development is still a debatable issue. Recently, inactivating mutations of hepatocyte nuclear factor-1a (HNF-1a) transcription factor gene or activating mutations of b-catenin have been demonstrated in some liver adenomas, and a possible link between HNF-1a gene mutations and oral contraceptives has been suggested. Only two cases of regressive LA after hormone withdrawal therapy have been described so far but without any information concerning the molecular characteristics of the tumours. Case: We report the case of a 48-year-old woman with LA, who had been taking an androgenic progestin therapy (lynestrenol) for 10 years. A major regression in the number and size of the lesions was observed 6 months after complete withdrawal of this therapy. Methods: Hepatocellular adenomas were studied by immunohistochemistry for oestrogen, progesterone and androgen receptors (ER, PR and AR respectively), and for b-catenin. Direct sequencing of the HNF-1a gene was also performed. Results: For the first time, we demonstrate significant Immunostaining of AR in the hepatocellular adenomas. This staining was negative in the partially regressive adenoma. Immunostainings for ER and PR were negative. HNF-1a and the b-catenin pathways were not involved in tumour pathogenesis. Conclusions: Our case suggests a role of androgenic progestin therapy in some cases of LA. Hormone therapy withdrawal may induce a significant regression in lesions.
Raoul Poupon - One of the best experts on this subject based on the ideXlab platform.
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reply to the sannier et al letter entitled mdr3 Immunostaining on frozen liver biopsy is not a sensitive diagnosis tool for the detection of heterozygous mdr3 abcb4 gene mutation
Virchows Archiv, 2012Co-Authors: Dominique Wendum, Veronique Barbu, Olivier Rosmorduc, Lionel Arrive, Jeanfrancois Flejou, Raoul PouponAbstract:Sir or Madam, We read with great interest the letter by Sannier et al. The data presented are consistent with our publication [1]. The authors studied a new series of adult patients with unexplained cholestasis and heterozygous MDR3/ABCB4 mutations. In five cases, a MDR3 Immunostaining was performed on liver frozen sections and showed an intense canalicular staining similar to a normal staining. These results are in contradiction with their initial report that showed a faint MDR3 Immunostaining in four patients with heterozygous MDR3/ABCB4 mutations [2]. This discrepancy may be explained by technical hazards or by the type of MDR3/ABCB4 mutations (indeed, the mutations were different in the two series). It could be interesting to perform MDR3 Immunostainings on the formalin-fixed paraffin-embedded liver tissues in these two series that would enable a comparison with our published data which were obtained with this technique. To conclude, it seems that MDR3/ABCB4 heterozygous mutation diagnosis requires gene sequencing.
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aspects of liver pathology in adult patients with mdr3 abcb4 gene mutations
Virchows Archiv, 2012Co-Authors: Dominique Wendum, Veronique Barbu, Olivier Rosmorduc, Lionel Arrive, Jeanfrancois Flejou, Raoul PouponAbstract:The aims of this study were to describe the histological liver lesions in adult patients with MDR3/ ABCB4 mutation and to study the usefulness of MDR3 Immunostaining as a diagnostic tool. All adult patients from our institution with an MDR3/ABCB4 mutation and a liver histology were included (n = 13). Eleven patients had a single heterozygous gene mutation and two patients had two heterozygous mutations. Two patients had no liver lesions. Eight patients had a mild ductular reaction and portal fibrosis. One patient had a few fibrous septa and two patients had biliary cirrhosis. In three cases intraductal lipid crystals were identified. Two patients had biliary fibroobliterative lesions with no sclerosing cholangitis on cholangiography. Biliary dysplasia was identified in hepatectomy specimens from two patients, one of whom developed an intrahepatic cholangiocarcinoma. One patient with biliary cirrhosis developed a hepatocellular carcinoma. MDR3 Immunostainings performed on formalin-fixed paraffin-embedded sections showed a strong canalicular staining in all patients except in one. To conclude, the predominant histological features were ductular reaction with no or mild fibrosis without cholangitis. Liver lesions previously unreported in association with MDR3/ABCB4 gene mutations (biliary dysplasia, cholangiocarcinoma, small duct sclerosing cholangitis) were also found. Lipid crystals in bile ducts may be suggestive of MDR3/ABCB4 mutation. MDR3 Immunostaining on formalin-fixed paraffin-embedded sections does not seem to be sensitive for the diagnosis of heterozygous MDR3/ABCB4 mutations.
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regressive liver adenomatosis following androgenic progestin therapy withdrawal a case report with a 10 year follow up and a molecular analysis
European Journal of Endocrinology, 2007Co-Authors: Magali Svrcek, Lionel Arrive, Jeanfrancois Flejou, Raoul Poupon, Emmanuelle Jeannot, Gaelle Fromont, Jessica Zucmanrossi, Philippe Bouchard, Dominique WendumAbstract:Objective: The relationship between sex hormones and hepatocellular adenoma development is well established. On the contrary, their contribution to liver adenomatosis (LA) development is still a debatable issue. Recently, inactivating mutations of hepatocyte nuclear factor-1a (HNF-1a) transcription factor gene or activating mutations of b-catenin have been demonstrated in some liver adenomas, and a possible link between HNF-1a gene mutations and oral contraceptives has been suggested. Only two cases of regressive LA after hormone withdrawal therapy have been described so far but without any information concerning the molecular characteristics of the tumours. Case: We report the case of a 48-year-old woman with LA, who had been taking an androgenic progestin therapy (lynestrenol) for 10 years. A major regression in the number and size of the lesions was observed 6 months after complete withdrawal of this therapy. Methods: Hepatocellular adenomas were studied by immunohistochemistry for oestrogen, progesterone and androgen receptors (ER, PR and AR respectively), and for b-catenin. Direct sequencing of the HNF-1a gene was also performed. Results: For the first time, we demonstrate significant Immunostaining of AR in the hepatocellular adenomas. This staining was negative in the partially regressive adenoma. Immunostainings for ER and PR were negative. HNF-1a and the b-catenin pathways were not involved in tumour pathogenesis. Conclusions: Our case suggests a role of androgenic progestin therapy in some cases of LA. Hormone therapy withdrawal may induce a significant regression in lesions.
Shan Rong Shi - One of the best experts on this subject based on the ideXlab platform.
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Comparative Study of Antigen Retrieval Heating Methods: Microwave, Microwave and Pressure Cooker, Autoclave, and Steamer
Biotechnic & histochemistry : official publication of the Biological Stain Commission, 1996Co-Authors: Clive R Taylor, Chen Chen, Christina Yang, Shan Rong Shi, Lillian L. Young, Richard J CoteAbstract:We present a study comparing the most popular heating methods currently used for antigen retrieval (AR) Immunostaining: the microwave oven, microwave with pressure cooker, autoclave, and steamer heating. A panel of 21 antibodies was tested on formalin fixed, paraffin embedded sections using these heating methods and Tris-HC1 buffer, pH 9.5, plus 5% urea as the AR solution. Three observers independently evaluated the intensity of AR Immunostaining. All heating methods yielded good results for AR Immunostaining. There were only minor differences among the heating methods for AR when the optimal concentration of primary antibody for normal Immunostaining was used; however, background staining may occasionally be troublesome if antibodies are not retitrated and diluted further for use on tissues after AR. Significant differences were observed only after further dilution of the primary antibodies: the microwave pressure cooker, extended microwave heating (5 min x 4) and autoclave heating then showed a similar intensity of staining that was stronger than results obtained with the steamer (20 min) or regular microwave heating (5 min x 2). Extension of the steamer heating time, however, yielded equivalent results. This study indicates that different heating methods can yield similar intensities of AR Immunostaining if the heating times are adjusted appropriately. It is noteworthy that, in general, the adjusted conditions for maximal retrieval differ from those most widely cited in the literature, or recommended by manufacturers. That several heating devices may provide similar results permits the use of different AR heating methods according to the equipment available. This study also is an early step in standardizing the AR Immunostaining protocol by providing uniform conditions for "maximal retrieval" as a common end point for all laboratories.
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antigen retrieval immunohistochemistry under the influence of ph using monoclonal antibodies
Journal of Histochemistry and Cytochemistry, 1995Co-Authors: Shan Rong Shi, Richard J Cote, Lillian Young, S A Imam, Clive R TaylorAbstract:Antigen retrieval (AR) incorporating high-temperature microwave (MW) heating of tissue sections before Immunostaining is a revolutionary technique that can unmask the antigens in formalin-fixed tissue sections, thus making them available for immunohistochemical staining. Although high temperature is believed to be the primary mechanism in retrieval of antigens, a variety of chemical solutions have been tested to define an optimal AR solution. We tested the hypothesis that pH of the AR solution may influence the quality of Immunostaining by using seven different AR buffer solutions at a series of different pH values ranging from 1 to 10. We evaluated the staining of monoclonal antibodies to cytoplasmic antigens (AE1, HMB45, NSE), nuclear antigens (MIB-1, PCNA, ER), and cell surface antigens (MT1, L26, EMA) on routinely formalin-fixed, paraffin-embedded sections under different pH conditions with MW heating for 10 min. The intensity of Immunostaining was graded in a blinded fashion. The pH value of the AR buffer solution was carefully measured before, immediately after, and 15 min after the AR procedure. The influence of pH on AR immunohistochemical staining can be summarized into three patterns. Some antigens (L26, PCNA, AE1, EMA, and NSE) showed excellent retrieval throughout the pH range. Other antigens (MIB1 and ER) showed strong intensity of immunohistochemical staining at very low pH and at neutral to high pH, but a dramatic decrease in the intensity of the AR Immunostaining at moderately acidic pH (pH 3-6). Still others (MT1 and HMB45) showed increasing intensity of the AR Immunostaining with increasing pH, but only weak Immunostaining at low pH. Among the seven buffer solutions at any given pH value, the intensity of AR Immunostaining was very similar. However, Tris-HCl buffer tended to produce better results at higher pH, compared with other buffers. Although high-temperature heating is believed to be the most important factor for the AR technique, the pH value of the AR solution is an important co-factor for some antigens. Optimization of the AR system should therefore include optimization of the pH of the AR solution. Our results indicate that AR Immunostaining of Tris-HCl or sodium acetate buffer at pH 8-9 may be suitable for most antigens, although certain nuclear antigens show optimal staining at low pH.
