Abstract
Simultaneous analyses of glycogen in sections with other subcellular constituents within the same section will provide detailed information on glycogen deposition 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 genuine glycogen values. Conventional PAS was successfully modified for combined use with immunofluorescence. Transmitted light microscopic examination of glycogen was successfully followed by semiquantification of glycogen using microdensitometry. Semiquantitative data correlated perfectly with glycogen content measured biochemically in the same sample (r2=0.993, P<0.001). Using a calibration curve (r2=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 semiquantitative 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.
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Acknowledgements
The antibodies referred to as 9D10, 2E8, N2.261 and A4.840 used in the present study were obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242, USA. The research of Dr. R.P. Hesselink was supported by a research grant from the Prinses Beatrix Fonds.
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Schaart, G., Hesselink, R.P., Keizer, H.A. et al. A modified PAS stain combined with immunofluorescence for quantitative analyses of glycogen in muscle sections. Histochem Cell Biol 122, 161–169 (2004). https://doi.org/10.1007/s00418-004-0690-0
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DOI: https://doi.org/10.1007/s00418-004-0690-0