Immunohistochemistry and immunocytochemistry (ICC) play an irreplaceable role in research and diagnostics. It is well known that antigen retrieval (AR) can, as a technique, have beneficial outcomes on immunohistochemistry results when using formalin-fixed, paraffin-embedded tissue samples. The main purpose of AR is to break protein crosslinks which are formed during formalin fixation. Although AR was originally designed for formalin-fixed, paraffin-embedded samples, the usefulness of AR in ICC has been described in previous studies. Cytologic samples are often fixed in alcohol-based fixatives which does not lead to the formation of crosslinks. Therefore, alcohol-fixed samples can be successfully immunostained without AR. We investigated the effect of heat-induced antigen retrieval (HIAR) on alcohol-fixed HEK293 cell line samples and patient cytologic samples from thyroid gland obtained by fine needle aspiration technique. We compared indirect 2-step ICC staining results performed according to the protocol with or without HIAR in citrate buffer pH 6 for several antibodies. Utilizing HIAR against intracellular antigens has beneficial effects. Therefore, more diluted antibodies can be used for satisfactory results. However, surface antigens were probably damaged by HIAR treatment. We demonstrated evident changes in cell surface topography after HIAR treatment by atomic force microscopy. Staining specificity of patient samples improves and background staining is reduced, allowing higher dilutions of primary antibody. Improving staining specificity is necessary for accurate diagnostics. Although we have shown the beneficial effect of HIAR for immunostaining intracellular antigens, proper staining protocol should be tested on appropriate controls for individual antibodies.

Biology

Departments of Histology and Embryology

Institute of Molecular and Translational Medicine

Laboratory of Molecular Pathology Department of Clinical and Molecular Pathology Faculty of Medicine and Dentistry Palacky University Olomouc Czech Republic

Medical Biophysics

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