THE MATPASE HISTOCHEMICAL PROFILE OF RAT TYPE-IIX FIBERS - CORRELATION WITH MYOSIN HEAVY-CHAIN IMMUNOLABELING

In the present study we report a novel histochemical method which, by sequential pre-incubations in alkaline and acidic media, selectively d ifferentiates muscle fibres expressing myosin heavy chain IIX, on the basis of a specific profile for myofibrillar actomyosin ATPase (mATPas e) activity. The enzyme reactions were tested for specificity by means of anti-myosin heavy chain monoclonal antibodies, which were characte rized on Western blots of muscle homogenates. Enzyme histochemical rea ctions with the traditional pH buffers were compared to those of the n ew method and, in conjunction with the immunoreactions, used to confir m the relationship between MyHC expression and the distinct profiles f or mATPase. Immunohistochemical reactions demonstrated that the new me thod only differentiates those fibres expressing myosin heavy chain II X. The method revealed a continuum in which the intermediate staining intensities corresponded to hybrid fibres expressing myosin heavy chai n IIX in combination with either the IIA or IIB forms. Quantitative hi stochemistry and immunohistochemistry (by image analysis), used to exa mine the relationship between staining intensities for mATPase and amo unts of myosin heavy chain IIX expression, revealed that the new metho d discriminates well between hybrid fibres expressing variable amounts of the IIX isoform (r(2) = 0.93).