Secondary reagents for immunohistochemical research of rat brain

Abstract

BACKGROUND: The critical factor when working with immunohistochemistry in laboratory animals is to select appropriate secondary antibodies, that allow clear and specific visualization of tissue antigens. Many reliable secondary reagents are currently not available for purchase, which determines the high relevance of replacing them with other detection systems. AIM: To verify the effectiveness of available secondary reagents for immunohistochemical research of the rat brain. MATERIALS AND METHODS: Brain samples from Wistar (n=2) and SHR (n=2) were used for the study. Iba-1, GFAP and vimentin immunohistochemistry was carried out using various polymer-based detection systems, namely UltraVision Quanto Detection System HR, N-Histofine Simple Stain MAX PO and UnoVue Rabbit HRP. RESULTS: All three studied polymer systems demonstrated visualisation of target proteins in brain tissues and cells corresponding to the general understanding of structures containing Iba-1, GFAP and vimentin. The UnoVue Rabbit HRP and UltraVision Quanto Detection System HRP kits showed good and similarly specific immunohistochemical reaction. However, the UnoVue Rabbit HRP kit was less sensitive compared to the UltraVision Quanto Detection System HRP. The reaction with N-Histofine Simple Stain MAX PO was not optimal due to the presence of non-specific background staining that was not present with other reagents. Apparently, this is due to the focus of the kit on immunohistochemical staining of human tissue and the likely absence of an antibody purification with rat serum. CONCLUSIONS: Two secondary antibody kits from the three studied showed optimal efficiency of immunohistochemical reaction and minimal background staining. N-Histofine Simple Stain MAX PO is not suitable for immunohistochemical research of rat brain tissue.