Endogenous biotin and background staining in murine cardiac tissue using the avidin-biotin immunoperaxidase technique
Creators: name: Ku, Wenwei
Publisher: name: University of Utah
Year: 2012
Subjects: subject: Immunology
subject: FOS: Clinical medicine

description: Previous investigations have shown that there is an increased expression of major histocompatibility complex (MHC) molecules during allograft rejection due to cytokines secreted by lymphocytes and monocytes following stimulation by alloantigens or viral infection. In order to investigate the expression of H-2 molecules in allografted cardiac tissue, we used a modified direct peroxidase staining method based on the avidin-biotin reaction.  Immunoperoxidase staining based on the avidin-biotin reaction is sensitive and widely used. This study reports the problems encountered when we applied this technique or staining fresh frozen BALB/c strain mouse heart tissue, the mechanisms responsible for the difficulties, as well as modifications to the procedures which eventually allowed successfiil staining and identification of MHC molecules on allograft tissue. The commercially available hybridoma cells that produce anti-H-2 K-d-D-d monoclonal antibodies needed to demonstrate MHC antigen on BALB/c tissue are generated by fiision of mouse myeloma cells and mouse spleen cells. In an indirect staining protocol, secondary antibodies to be used are customarily elicited by injecting mouse IgG into animals of a different species, such as rabbit or goat; the resultant anti-mouse IgG antibody will tend to react not only with the specific primary IgG attached to target cells, but also to any IgG already present in the target prior to application of the primary antibody. Therefore, a false positive staining may result because of the same species origin of primary antibodies generated in the tissue to be stained. Furthermore, in a direct staining procedure which avoided the secondary antibody problem described above, the reagent avidin-horseradish-peroxidase also caused excessive staining. To avoid the application of mouse antibody to mouse tissue in the indirect staining method, a modified direct staining method involving biotinylated anti-H-2 monoclonal antibodies was used. Endogenous biotin in mouse heart tissue was found to be a major cause of excessive staining in the avidin-biotin based immunoperoxidase staining technique. It was overcome by pretreatment of the tissue with free avidin solution and followed by free biotin solution. The free avidin combined with endogenous biotin, and the free biotin applied thereafter saturated any remaining avidin binding sites.  Normal inbred mouse BALB/c heart tissue treated with anti-H-2 K-d-D-d antibody showed little staining of myocyte plasma and intercalated discs, but definitive staining of nuclear membranes, myocyte membranes and the endothelium of arterioles and capillaries. Syngeneically grafted BALB/c inbred mouse heart tissue given the same treatment showed staining similar to that of a normal BALB/c heart. In contrast, allografted heart tissue, harvested and stained day 4 posttransplantation, showed increased staining on myocyte membranes and discs and even stronger staining of nuclear membranes and endothelium when compared to normal BALB/c mouse heart tissue.  The regulation of MHC molecule expression on allogeneic cardiac grafts, the role of cytokines, and the potential effect of murine cytomegalovirus (MCMV) infection on MHC molecule expression are also reviewed and discussed. Further studies will be needed to determine the expression of MHC Class II during allograft rejection, and also the relationship between cytokine levels and MCMV infection in regard to MHC molecule expression on murine allogeneic heart tissue using the described procedures.