Step-by-step protocol for chromogenic immunohistochemical staining of paraffin sections of animal organs in viral infections, using the example of SARS-CoV-2

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Abstract
Introduction. Immunohistochemical (IHC) analysis is one of the main methods for detecting and visualizing the protein determinants of the virus in organs. The nanoscale structures of viruses complicate the visualization of particle topography, which prevents a more detailed understanding of the pathogenesis of viral infections. Unfortunately, general IHC protocols are not universal and often their application in special cases is impossible. Optimizing the coloring methodology can significantly save research time. Based on this, this paper describes a step-by-step methodology for IHC staining of paraffin sections of animal organs infected with the SARS-CoV-2 virus. The purpose of the study. Optimization of the chromogenic IHC staining protocol when working with infected material, using the example of SARS-CoV-2. Materials and methods. IHC analysis was performed on paraffin sections of lung tissue of ferrets (Mustela putorius furo) infected with Omicron and Delta strains of SARS-CoV-2. Results. During the study, it was demonstrated that the optimal exposure time for samples with hydrogen peroxide was at least 30 minutes. The use of BSA before the application of primary antibodies also contributed to a decrease in background staining, while the optimal concentration of the reagent was at least 1%. It is noteworthy that the addition of BSA to the primary antibody solution also contributed to a decrease in the non-specific signal. In the context of the current work, dilution of antibodies applied to S-protein by 1000 times was optimal to minimize the proportion of non-specific tissue staining. Conclusion. In the work on optimizing the IHC protocol for paraffin sections, the main aspects affecting the staining quality were demonstrated and described. When working with material containing SARS-CoV-2, options for eliminating methodological mistakes were proposed.

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