Quantitative histochemical investigations of semipermeable membrane techniques for the assay of acid phosphatase in skeletal muscle. IV. A post-coupling technique.
A post-coupling semipermeable membrane technique for determining the activity of acid phosphatase in sections of skeletal muscle has been developed and investigated for its reproducibility and validity. Cryostat sections of unfixed muscle mounted on dry dialysis membranes are first incubated for 1-4 at 37 degrees C on a gelled medium containing 4 mM naphthol AS-BI phosphate buffered at pH 5. They are next transferred to another gel containing only hexazotised Pararosanaline and incubated for a further 30 min. Finally, they are treated with 70% ethanol, dried in air, and mounted. The final reaction product (FRP) deposited within muscle fibres is mostly distributed as a fine reticular network, tentatively identified as sarcoplasmic reticulum. Large FRP "granules' of the kind observed with Meijer's simultaneous coupling membrane technique are not formed. The method is reproducible and valid in terms of several working criteria. For example, the mean absorbance of the FRP at its absorption maximum increases linearly with incubation time; and in model sections containing various amounts of a subcellular fraction rich in acid phosphatase, the mean absorbance after a constant incubation time is proportional to the enzyme concentration. FRP is formed at approximately twice the rate it is deposited in the simultaneous coupling method. The most important advantage of the post-coupling method over the simultaneous coupling method is that the inhibition of the enzyme by coupling reagents is avoided.