Glycogen determination using periodic acid-schiff: artifact of muscle preparation.
OBJECTIVE
It is common practice for the staining of muscle glycogen with periodic acid-Schiff (PAS) to thaw and dry muscle sections before staining. The goal is to investigate whether this thawing step results in a systematic error that is independent of muscle fiber type and muscle physiological state.


METHODS
Muscle samples from six fasted male subjects were obtained before or after 3 min of high-intensity cycling. Each sample was sectioned; some sections were assessed for muscle fiber composition, and others were either thawed for 20 min or kept frozen before being PAS-stained for glycogen. The response to a 20-min freeze-thaw cycle was also assessed using rested and exercised rats as our experimental model, and the changes in glycogen were measured enzymatically.


RESULTS
The inclusion of a 20-min thawing step resulted in a significant reduction (P < 0.05) in the weighted average of the optical density of PAS (ODPAS) staining in both the nonexercised (15 +/- 1.4%) and exercised human muscles (15 +/- 1.3%), with the absolute extent being greater in the nonexercised muscle samples (P < 0.05). Moreover, the observed decrease in ODPAS was greatest in Type IIa fibers for both the nonexercised (P < 0.05) and exercised (P < 0.05) muscle samples. The findings in rats suggest that the muscle damage associated with freeze-thawing is responsible for this stimulation of glycogenolysis.


CONCLUSIONS
For the quantitative histochemical measurement of glycogen content in skeletal muscle, the common practice of thawing unfixed muscle sections before PAS staining should be abandoned because this causes glycogen breakdown, the extent of which varies across muscle fiber types and prior exercise history.
Fairchild
Timothy J
TJ
School of Human Movement and Exercise Science, University of Western Australia, Crawley, Western Australia, Australia 6009. timf@cyllene.uwa.edu.au
Fournier
Paul A
PA
D000328:Adult; D016477:Artifacts; D015925:Cryopreservation; D015444:Exercise / Q000502:physiology; D006003:Glycogen / Q000032:analysis*; D006801:Humans; D008297:Male; D018482:Muscle, Skeletal / Q000737:chemistry*; D010503:Periodic Acid-Schiff Reaction; D015203:Reproducibility of Results; D013048:Specimen Handling
D006003:Glycogen