A new immunohistochemical method for quantification of fast-myosin in frozen histologic sections of the rat skeletal muscles.

An immunohistochemical micromethod for quantification of fast-myosin in frozen histologic sections of rat skeletal muscles was developed. The principle of this method was enzyme-linked immunosorbent assay (ELISA). We used frozen tissue sections as models of the antigen-coated wells of ELISA plate. The intensity of immunoreactivity of the frozen section to an anti-fast-myosin monoclonal antibody was quantified directly from the color developed with the second antibody coupled with peroxidase using phenol-4-aminoantipyrine as a substrate. Then, the same section was incubated in 0.01 M acetic acid solution to cleave antigen-antibody complexes, followed by colorimetric measurement to obtain the absolute value of total protein per section. Fast-myosin content in the frozen tissue section was expressed as mg of fast-myosin per g of total protein. In this micromethod, the minimum area and the optimum thickness of the section were 5 mm2 and 10 microns, respectively. Fast-myosin contents in the extensor digitorum longus and soleus muscles were 185.0 +/- 6.1 and 17.5 +/- 2.4 mg/g, respectively. The results obtained by this micromethod were in good agreement with those obtained by two conventional methods, immunohistochemical morphometry and biochemical determination. This micromethod is useful for a quantitative evaluation of the contractile function of the mammalian skeletal muscles.