Demonstration of myoglobin and CK-M in myocardium. Comparison of five fixation methods and three immunohistochemical techniques.

The results of immunohistochemical staining vary depending on the tissue, fixative, antigen-antibody system, and immunohistochemical staining methods used. The purpose of this study was to evaluate the effect of different methods of fixation, different antigen-antibody systems, and different immunohistochemical methods on immunohistochemical staining of myocardium. Samples of normal fresh canine myocardium from six dogs were fresh frozen and fixed in 10% neutral buffered formalin, Bouin's, Bayley's and Carnoy's fixatives. Immunohistochemical staining for myoglobin and creatine kinase M was performed using the ABC (avidin-biotin complex) and indirect peroxidase-antiperoxidase (PAP) techniques. Tissues fixed in formalin showed the most intense specific staining for both antigens with the least background and nonspecific staining. All other fixation methods and frozen section techniques gave a more variable degree of specific positive staining and substantial background staining and/or nonspecific staining. ABC and PAP techniques gave similar results with both antigen-antibody systems and with each fixation method. Thus, no differences in specificity or sensitivity were observed between ABC and PAP techniques. Differences in staining intensity and pattern were related primarily to differences in fixation methods.