ABSTRACT
Objective: The study aimed to find the best antigen retrieval buffer and heat source for the immunohistochemical staining of various kinds of antibodies in our laboratory.Materials and methods: We designed the method to evaluate the efficacy of three different retrieval solutions including 10mM Tris-HCl + 1mM EDTA, pH 9, 0.05%citraconic anhydride solution pH 7.4 and 10 mM citrate buffer pH 6, and 3 heat source pretreatment methods (Microwave, pressure cooker and water bath treatment)to retrieve twenty-one immunoreactivity in formalin-fixed, paraffin-embedded sections.Results: We found that, modified retrieval solution, 10mM Tris-HCl + 1mM EDTA buffer, pH 9 is the most efficient for a large variety of antibodies and not depending on heat sources. On the other hand, 0.05% citraconic anhydride solution and 10mM citrate buffer are moderate and poor retrieval solutions, respectively. Moreover, these two solutions are heat source-dependent.Conclusion: These results demonstrate that 10mM Tris-HCl + 1mM EDTA, pH 9 and heat-pretreatment is useful for the immunohistochemistry of many antigens in aldehyde-fixed, paraffin-embedded tissues. Keywords: antigen retrieval, immunohistochemistry, citraconic anhydride, formaldehyde
ABSTRACT
The expression of candidate genes in tissue samples were investigated by using immunohistochemical study of formalin-fixed paraffin-embedded (FFPE) tissue or immunofluorescence labeling of cryosections. However, the study of co-localization of multiple antigens in the same section by immunohistochemical labeling is difficult to evaluate and involves antigen retrieval step to unmask antigen whereas immunofluorescence labeling has the capability for multiple labeling with higher resolution. Nevertheless, the handling of fresh tissue as immunofluorescence labeling is difficult. Thus, both methods have limitations as research tools. In this study, we optimized an antigen retrieval method for high-resolution immunofluorescence labeling of FFPE invasive breast cancers using three different biomarkers (Ki-67, Her-2 protein and E-cadherin). Citraconic anhydride solution at 0.05 % pH 7.4 in a microwave oven seemed to work well for multiple antibodies for co-localization of multiple antigens in the same section. In addition, this method is suitable for core needle biopsy which obtained small amount of tissue. Therefore, it is not enough for preparation of multiple sections.