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Comparison of Different Methodologies and Cryostat versus Paraffin Sections for Chromogenic Immunohistochemistry
Author(s) -
Hira Vashendriya,
Jong Annique Loncq,
Ferro Klea,
Khurshed Mohammed,
Molenaar Remco,
Van Noorden Cornelis J.F.
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.802.3
Subject(s) - antigen retrieval , primary and secondary antibodies , antigen , immunohistochemistry , horseradish peroxidase , microbiology and biotechnology , chemistry , antibody , tonsil , staining , antigenicity , pathology , biology , immunology , biochemistry , medicine , enzyme
Immunohistochemistry (IHC) specifically localizes proteins in cells and tissues, but methodologies vary widely. Therefore, we performed a methodological IHC optimization and validation study. First, we compared advantages and disadvantages of cryostat sections versus paraffin sections. Second, we compared and optimized antigen retrieval in paraffin sections using citrate buffer and Tris/EDTA buffer. Third, aminoethyl carbazole (AEC) and 3,3′‐diaminobenzidine (DAB) were tested as horseradish peroxidase (HRP) substrates to obtain a water‐insoluble coloured end product to visualize antigens. Fourth, secondary antibodies conjugated with either mono‐HRP or poly‐HRP were compared. The study was performed using serial sections of human tonsil. IHC was performed with primary antibodies against endothelial cell marker CD31, smooth muscle actin (SMA), chemokine stromal‐derived factor‐1α (SDF‐1α) and its receptor C‐X‐C receptor type 4 (CXCR4), macrophage marker CD68 and proliferation marker Ki67. DAB rather than AEC, and cryostat sections rather than paraffin sections gave optimum staining at highest primary antibody dilutions, whereas tissue morphology in paraffin sections was superior. Loss of antigenicity in paraffin sections by formaldehyde fixation, heat and/or masking of epitopes was counteracted by antigen retrieval but not for all antigens. Two out of six antigens (CD31 and CD68) could not be retrieved irrespective time and type of retrieval. Tris‐EDTA was superior to citrate buffer for antigen retrieval. The use of mono‐HRP or poly‐HRP depended on the affinity of the primary antibody for its antigen. We conclude that IHC methodology optimization and validation are crucial steps for each antibody and each research question. Support or Funding Information KWF; UVA 2014‐6839 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .