A modified PAS stain combined with immunofluorescence for quantitative analyses of glycogen in muscle sections.
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.
Department of Movement Sciences, Nutrition and Toxicology Research Institute Maastricht, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands.
Gert
G
Schaart
Reinout P
RP
Hesselink
Hans A
HA
Keizer
Gerrit
G
van Kranenburg
Maarten R
MR
Drost
Matthijs K C
MK
Hesselink
D000328:Adult; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D002452:Cell Count; D004396:Coloring Agents; D003599:Cytoskeleton / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006003:Glycogen / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007074:Immunoglobulin G / Q000737:chemistry; D007382:Intermediate Filaments / Q000378:metabolism; D007536:Isomerism; D008297:Male; D018482:Muscle, Skeletal / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000378:metabolism; D010503:Periodic Acid-Schiff Reaction; D051381:Rats; D017208:Rats, Wistar; D012015:Reference Standards; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism
D000906:Antibodies; D004396:Coloring Agents; D007074:Immunoglobulin G; D006003:Glycogen; D018995:Myosin Heavy Chains