Cytochemical staining procedures selective for sarcotubular systems of muscle: Modifications and applications

Abstract

The diaminobenzidine (DAB)-ferrocyanide cytochemical procedure for staining of myocardial sarcoplasmic reticulum (SR), first described by Waugh and his co-workers, has been subjected to further experimentation and modification. We have found, on the basis of studies utilizing inhibitory substances and conditions, that the staining reaction in the SR apparently is not attributable to endogenous peroxidase activity within the lumina of the SR tubules and saccules (nor, for that matter, to the activity of any species of enzyme). Perfusion of the vascular system with DAB-H2O2-Tris solution is unnecessary. In fact, exposure of tissues to DAB itself is superfluous, since mere postfixation in osmium—ferrocyanide solution serves to produce SR staining in muscle cells that have been fixed in an aldehyde solution containing calcium (or other divalent or trivalent cations), but lacking phosphate. This technique is applicable to both cardiac and skeletal muscle cells of mammals and lower vertebrates; it has been applied with success to mammalian vascular and nonvascular smooth muscle as well. Some degree of filling (or staining) of the system of extracellular spaces [ECS, including, in mammalian myocardium, the T-axial tubular system (TAx)] often occurs, which appears to be the result of colloid formation by the osmium ferrocyanide. SR staining in skeletal muscle usually is inseparable from T-system filling, although filling of T tubules may occur in the absence of SR staining. Choice of concentration or type of aldehyde(s) in the fixative solution does not appreciably alter the staining; however, exposure of tissues to aldehyde fixative which either does not contain calcium or contains phosphate brings about filling of the ECS-TAx without SR staining, thereby mimicking in effect the results obtained by more complicated tracer techniques.