Related Experiment Video
Updated: Aug 13, 2026
Biochemical Titration of Glycogen In vitro
Published on: November 25, 2013
Glycogen determination using periodic acid-schiff: artifact of muscle preparation
Timothy J Fairchild1, Paul A Fournier
1School of Human Movement and Exercise Science, University of Western Australia, Crawley, Western Australia, Australia 6009. timf@cyllene.uwa.edu.au
Purpose:
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.
Conclusion:
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.
Insights
Thawing muscle sections before periodic acid-Schiff (PAS) staining significantly reduces glycogen content, especially in Type IIa fibers. This common practice introduces systematic error and should be avoided for accurate muscle glycogen measurement.
Area of Science:
- Muscle physiology
- Histochemistry
- Biochemistry
Background:
- Muscle glycogen is crucial for energy metabolism.
- Periodic acid-Schiff (PAS) staining is a common method for quantifying muscle glycogen.
- Current protocols often involve thawing muscle sections before staining.
Purpose of the Study:
- To determine if thawing muscle sections before PAS staining introduces systematic error.
- To investigate if this error is independent of muscle fiber type and physiological state.
Main Methods:
- Muscle samples from human subjects and rats were analyzed.
- Sections were either thawed or kept frozen before PAS staining.
- Glycogen content was measured using optical density and enzymatic assays.
Main Results:
- A 20-minute thawing step significantly reduced PAS staining intensity in both rested and exercised human muscle (15%).
- Glycogen reduction was more pronounced in Type IIa fibers.
- Experiments in rats indicated freeze-thaw damage stimulates glycogenolysis.
Conclusions:
- Thawing unfixed muscle sections before PAS staining causes glycogen breakdown.
- This breakdown introduces quantitative errors in glycogen measurement.
- The practice should be abandoned to ensure accurate glycogen analysis in skeletal muscle.
