Localization of Myoglobin in Striated Muscle of the Domestic Pig; Benzidine and NADH2-TR Reactions

Tissue blocks 1 cm³ from longissimus (white) and trapezius (red) muscles of adult pigs were fixed in phosphate-buffered 2.5% glutaraldehyde, pH 7.4, for 4 hr at about 25 C; washed 4 hr in running tap water, and immersed in 30% w/v sucrose solution for 16 hr or more. After freezing in liquid N₂, cryostat sections were cut and floated into saturated aqueous benzidine containing 0.15% H₂O₂ at 25 C for 30 min. Stained sections were washed in distilled water and mounted on slides with glycerol jelly. Three distinguishable gradiations of color intensity were found: strong, intermediate, and negative. The trapezius had a greater number of myoglobin-positive fibers than the longissimus muscle. Myoglobin-positive and myoglobin-negative staining occurred in red and white fibers, respectively; intermediates were apparently more closely related to the red than to the white fibers. The NADH₂TR reaction showed the same sites as did the benzidine reaction.