Identification of microglia and macrophages using antibodies to various sequences of the Iba-1 protein

Abstract

The Iba-1 protein is traditionally considered a highly selective marker of microglia because of the specific expression of the gene in this particular population of the CNS cells. Alternative splicing creates several isoforms of the Iba-1 protein, which may cause discrepancies in the results of immunohistochomic reactions depending on which epitopes of the immunogen the antibodies selected for the study were developed. In this connection, and with the aim at identifying reliable variants of antibodies to Iba-1 available to researchers in the Russian Federation, we organized with study, seeking to evaluate the results of detecting microglia and macrophages using antibodies to different protein sequences produced by different manufacturers. As material, we used samples of the brain and testis of sexually mature (3–5 months) male Wistar rats (n = 8). Polyclonal and monoclonal (clone JM36-62) antibodies to Iba-1 were used as primary reagents. We found that monoclonal antibodies of the JM36-62 clone enable more selective antigen detection with a better signal/background ratio; they can be used as replacements for reagents that are currently not available commercially. Polyclonal antibodies enabled not only immunospecific imaging of microglia and macrophages, but also the identification of cells of the epithelial-spermatogenic layer of the testis. It is assumed that epithelial-spermatogenic layer contains the Iba-1 isoform devoid of an epitope that corresponds to the sequence of the immunogenic antibody clone JM36-62 fragment of the native protein. Functionally, various isoforms of Iba-1 should be investigated further.