Histochem Cell Biol (2004) 122:161–169 DOI 10.1007/s00418-004-0690-0 ORIGINAL PAPER Gert Schaart · Reinout P. Hesselink · Hans A. Keizer · Gerrit van Kranenburg · Maarten R. Drost · Matthijs K. C. Hesselink A modified PAS stain combined with immunofluorescence for quantitative analyses of glycogen in muscle sections Accepted: 5 July 2004 / Published online: 3 August 2004  Springer-Verlag 2004 Abstract Simultaneous analyses of glycogen in sections with other subcellular constituents within the same sec- tion will provide detailed information on glycogen de- position and the processes involved. To date, staining protocols for quantitative glycogen analyses together with immunofluorescence in the same section are lacking. We aimed to: (1) optimise PAS staining for combination with immunofluorescence, (2) perform quantitative glycogen analyses in tissue sections, (3) evaluate the effect of section thickness on PAS-derived data and (4) examine if semiquantitative glycogen data were convertible to gen- uine glycogen values. Conventional PAS was successfully modified for combined use with immunofluorescence. Transmitted light microscopic examination of glycogen was successfully followed by semiquantification of gly- cogen using microdensitometry. Semiquantitative data correlated perfectly with glycogen content measured biochemically in the same sample (r 2 =0.993, P<0.001). Using a calibration curve (r 2 =0.945, P<0.001) derived from a custom-made external standard with incremental glycogen content, we converted the semiquantitative data to genuine glycogen values. The converted semiquanti- tative data were comparable with the glycogen values assessed biochemically (P=0.786). In addition we showed that for valid comparison of glycogen content between sections, thickness should remain constant. In conclusion, the novel protocol permits the combined use of PAS with immunofluorescence and shows valid conversion of data obtained by microdensitometry to genuine glycogen data. Keywords PAS · Glycogen · Immunofluorescence · Quantification · Muscle Introduction Excess dietary carbohydrates can be stored as the glucose polymer glycogen. In humans and rodents the vast ma- jority of glycogen is stored in liver and skeletal muscle. In healthy skeletal muscle, glycogen is dispersed in granules that can be found distributed as intra- and in- termyofibrillar granules (Marchand et al. 2002). In ad- dition, glycogen content varies considerably among dif- ferent muscle fibre types (Baldwin et al. 1977). Under some pathological conditions, glycogen storage or deg- radation is affected resulting in excessive storage of glycogen within the myocyte, which in turn interferes with contractile and metabolic performance of the af- fected myocytes. One such pathological condition is glycogen storage disease type II (GSDII), also referred to as Pompe’s disease. In GSDII, large amounts of glycogen are stored in lysosomes due to the lack of 1,4-a-gluco- sidase, the enzyme essential for hydrolysis of the 1,4- linkage in glycogen. Patients suffering from this disease are characterised by severe muscle wasting and muscle weakness (DiMauro and Lamperti 2001). In a mouse model mimicking GSDII (GSDII mice), the gene en- coding for 1,4-a-glucosidase has been knocked out, giving rise to a pathology very similar to GSDII, in- cluding lysosomal glycogen accumulation and muscle weakness (Bijvoet et al. 1999; Hesselink et al. 2002), with an increased number of apoptotic nuclei as a puta- tively important player (Hesselink et al. 2003). Histo- logical examination of the subcellular storage sites and the abundance of muscle glycogen, together with (im- muno)fluorescence microscopy to examine localisation and expression of many distinct proteins, may turn out to be a valuable, however presently unavailable, tool to study this hypothesis. Here, we aim to combine (quantitative) histological examination of muscle glycogen with immunodetectable proteins within the same section. Routinely, the presence of glycogen can be examined histochemically in cryo- sections by the periodic acid-Schiff (PAS) reaction, originally described by MacManus (1948). After on-sec- G. Schaart · R. P. Hesselink · H. A. Keizer · G. van Kranenburg · M. R. Drost · M. K. C. Hesselink ( ) ) Department of Movement Sciences, Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands e-mail: Matthijs.Hesselink@BW.unimaas.nl Tel.: +31-43-3881317 Fax: +31-43-3670972