KINETIC MICROPHOTOMETRIC EVALUATION OF IN-SITU HYBRIDIZATION FOR MESSENGER-RNA OF SLOW MYOSIN HEAVY-CHAIN IN TYPE-I AND C-FIBERS OF RABBIT MUSCLE

The present study was undertaken in order to test the possibility of m icrophotometric evaluation of in situ hybridizations. The histochemica l detection of mRNA specific to the slow myosin heavy chain (HCI), in fibre cross sections of normal and transforming rabbit muscles with a digoxigenin-labelled complementary RNA (cRNA) probe was used as a mode l. Scanning densitometry of Northern blot hybridizations showed that t he detection of cRNA/mRNA hybrids by a staining reaction catalysed by alkaline phosphatase coupled to an anti-digoxigenin antibody occurs in a concentration-dependent manner and follows a linear time course. Th ese findings were the basis for elaborating a comparative microphotome tric evaluation of in situ hybridization in tis sue sections by measur ing the reaction rate of the alkaline phosphatase-catalysed formazan p roduction. Relative amounts of HCI mRNA were thus determined by compar ing reaction rates instead of by single point microphotometry. This me thod was applied to studies on the distribution of HCI mRNA in differe nt fibre types of normal rabbit muscles and and muscles undergoing fas t-to-slow fibre transformation in response to low-frequency stimulatio n. The different fibre types were identified by histochemical staining for myofibrillar actomyosin ATPase (mATPase) in cross sections adjace nt to the sections processed for in situ hybridization. On the average , type I fibres displayed 2.3-fold higher reaction rates than the mean value recorded far C fibres. According to the pronounced scattering o f the values measured in single C fibres, these fibres represented a h eterogeneous population in the transforming muscle. Finally, results o btained from low-frequency stimulated muscle indicated that fibres whi ch were still unambigously identified as type II, started to express l ow amounts of HCI mRNA.