LOCALIZATION OF SKELETAL MUSCLE PHOSPHORYLASE USING A FLUORESCENT ANTIBODY TECHNIQUE AND ITS CORRELATION WITH HISTOCHEMICAL OBSERVATIONS

Abstract

The Coons fluorescent antibody technique was employed to localize rabbit muscle phosphorylase within striated muscle cells. Pooled sera from guinea pigs sensitized against 2X crystallized rabbit phosphorylase a in complete Freund's adjuvant gave a single line of precipitation with the enzyme in Ouchterlony plates. Such sera, tagged with fluorescein isothiocyanate and applied to thin frozen sections of rabbit striated muscle, produced a specific, granular, fluorescing precipitate which was localized to the sarcoplasm between myofibrils. Preincubation with untagged specific antisera prevented specific staining. Appropriate control sera, similarly tagged, produced only slight nonspecific fluorescence. With properly absorbed specific antisera, staining was specific for striated muscle as opposed to rabbit liver or smooth muscle. The fluorescent antibody findings were correlated with those obtained by classical histochemieal studies of phosphorylase activity. In addition, application of antisera to sections of rabbit muscle before incubation in substrate resulted in significant inhibition of histochemically demonstrable phosphorylase activity whereas control sera had little or no inhibitory effect.