A NEW IMMUNOHISTOCHEMICAL METHOD FOR QUANTIFICATION OF FAST-MYOSIN INFROZEN HISTOLOGIC SECTIONS OF THE RAT SKELETAL-MUSCLES

An immunohistochemical micromethod for quantification of fast-myosin i n frozen histologic sections of rat skeletal muscles was developed. Th e principle of this method was enzyme-linked immunosorbent assay (ELIS A). We used frozen tissue sections as models of the antigen-coated wel ls of ELISA plate. The intensity of immunoreactivity of the frozen sec tion to an anti-fast-myosin monoclonal antibody was quantified directl y from the color developed with the second antibody coupled with perox idase using phenol-4-aminoantipyrine as a substrate. Then, the same se ction was incubated in 0.01 M acetic acid solution to cleave antigen-a ntibody complexes, followed by colorimetric measurement to obtain the absolute value of total protein per section. Fast-myosin content in th e frozen tissue section was expressed as mg of fast-myosin per g of to tal protein. I this micromethod, the minimum area and the optimum thic kness of the section were 5 mm2 and 10 mum, 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 co nventional methods, immunohistochemical morphometry and biochemical de termination. This micromethod is useful for a quantitative evaluation of the contractile function of the mammalian skeletal muscles.