20.500.12556/DiRROS-19588 Comparison of different methodologies and cryostat versus paraffin sections for chromogenic immunohistochemistry 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. immunohistochemistry chromogens aminoethyl carbazole AEC 3 3'-diaminobenzidine DAB antigen retrieval tonsil true false true Angleški jezik Ni določen Neznano 2024-07-20 04:02:06 2024-07-24 09:15:18 2024-07-25 04:14:44 0000-00-00 00:00:00 2019 0 0 str. 125-134 iss. 2 Vol. 121 Feb. 2019 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 2019-02-01 577 0065-1281 10.1016/j.acthis.2018.10.011 5186895 RAZ_Hira_Vashendriya_V._V._2019.pdf RAZ_Hira_Vashendriya_V._V._2019.pdf 1 9CA91EABCFFFFFBC063B3242BBB85421 82aa90362ce69acfa056dea63b6ac188f92671821be2c9a959f1f1d1fac918c1 0ceaa64d-497c-11ef-b487-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=27249 https://doi.org/10.1016/j.acthis.2018.10.011 0 f5d6d469-497b-11ef-b487-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=27248 Nacionalni inštitut za biologijo 0 0 0