Quantitative histochemical investigations of semipermeable membrane techniques for the assay of acid phosphatase in skeletal muscle

Summary The reproducibility, specificity and validity of Meijer's semipermeable membrane simultaneous coupling technique for the assay of acid phosphatase activity in sections of skeletal muscle have been investigated quantitatively, using naphthol AS-BI phosphate as the substrate and hexazotised Pararosanaline (HPRA) as the coupler. With this technique, unlike conventional techniques, presumed specific final reaction product (FRP) is evident in three different histological sites in normal skeletal muscle; first, as intensely coloured red granules within muscle fibres; second, as a diffuse reddish colouration throughout the sarcoplasm of all muscle fibres (the intrafibre areas); and third, in certain connective tissue elements between the muscle fibres (the interfibre areas). The mean absorbance of the FRP (at its absorption maximum, 530 nm) formed in each of these sites after a constant incubation time does not differ significantly in serial sections. 6 mM sodium molybdate, an acid phosphatase inhibitor, reduces the mean absorbance by 50% in the intrafibre areas, but in the interfibre connective tissue areas, 1 mM is sufficient. In contrast, 10 mM EDTA, an alkaline phosphatase inhibitor, has a negligible effect on the formation of specific FRP. Thus, Meijer's technique appears to be reproducible and specific. The mean absorbance of the FRP formed in each of the three reactive histological areas increases linearly with incubation time and section thickness. The maximum amount of FRP is formed at pH 5 and when the substrate concentration is above about 4 mM. However, some of the FRP in the intrafibre areas is unspecific, and arises from the transformation of adsorbed HPRA to a purple-coloured product having an absorption maximum at 570 nm. Much of the non-specific FRP appears after the incubation has been terminated with formalin, and reaches a maximum several hours after the sections have been subsequently mounted. As a consequence, Meijer's technique is not entirely valid.