Anthony S-Y Leong1, Zenobia Haffajee. 1. Immunohistology Unit, Hunter Area Pathology Service and University of Newcastle, Newcastle, New South Wales, Australia. aleong@mail.newcastle.edu.au
Abstract
BACKGROUND: The introduction of heat-induced antigen retrieval has been a major milestone in diagnostic immunohistochemistry, enabling the application of many antibodies to fixed paraffin-embedded tissue sections. A number of important variables affect the preservation of tissue antigens, among which are analytical variables including the antigen retrieval methodology. Temperature of retrieval, duration of heating, source of heat, pH and nature of retrieval solution are among more important variables pivotal to results. Citrate buffer at pH 6.0 has become widely embraced as the universal fixative but some antibodies remain capricious and yield poor staining results. METHODS: This study examines the recent suggestion that citraconic anhydride may be a suitable universal retrieval reagent. Immunostaining of 65 commonly employed antibodies following microwave antigen retrieval in 0.05% citraconic anhydride for 10 minutes at 98 degrees C was compared with consecutive tissues sections subjected to antigen retrieval in citrate buffer at pH 6.0 at the same duration and temperature. RESULTS: Thirty-five of the 65 antibodies examined yielded more intense staining following antigen retrieval in citraconic anhydride, including some capricious antibodies such as MyoD1, myogenin, perforin, TIA-1, Tdt, RET and MiTF, confirming the efficacy of this retrieval solution. CONCLUSION: It is recommended that consideration be given to 0.05% citraconic anhydride as an antigen retrieval solution, particularly for antibodies that fail to work or stain weakly in fixed paraffin-embedded tissue sections.
BACKGROUND: The introduction of heat-induced antigen retrieval has been a major milestone in diagnostic immunohistochemistry, enabling the application of many antibodies to fixed paraffin-embedded tissue sections. A number of important variables affect the preservation of tissue antigens, among which are analytical variables including the antigen retrieval methodology. Temperature of retrieval, duration of heating, source of heat, pH and nature of retrieval solution are among more important variables pivotal to results. Citrate buffer at pH 6.0 has become widely embraced as the universal fixative but some antibodies remain capricious and yield poor staining results. METHODS: This study examines the recent suggestion that citraconic anhydride may be a suitable universal retrieval reagent. Immunostaining of 65 commonly employed antibodies following microwave antigen retrieval in 0.05% citraconic anhydride for 10 minutes at 98 degrees C was compared with consecutive tissues sections subjected to antigen retrieval in citrate buffer at pH 6.0 at the same duration and temperature. RESULTS: Thirty-five of the 65 antibodies examined yielded more intense staining following antigen retrieval in citraconic anhydride, including some capricious antibodies such as MyoD1, myogenin, perforin, TIA-1, Tdt, RET and MiTF, confirming the efficacy of this retrieval solution. CONCLUSION: It is recommended that consideration be given to 0.05% citraconic anhydride as an antigen retrieval solution, particularly for antibodies that fail to work or stain weakly in fixed paraffin-embedded tissue sections.
Authors: K Moriguchi; Y Mitamura; J Iwami; Y Hasegawa; N Higuchi; Y Murakami; H Maeda; F Yoshimura; H Nakamura; N Ohno Journal: Biotech Histochem Date: 2012-09-18 Impact factor: 1.718