Quantifying GSK3β in Muscle Fiber Types

To assess the fiber type expression of GSK3β, we performed immunofluorescent microscopy experiments on serial 10 μm cryosections obtained from O.C.T. (optimal cutting temperature) embedded soleus and EDL muscles from n = 3 mobile wild-type C57BL/6J male mice. One serial cryosection underwent immunofluorescent fiber type staining as previously described to demarcate the various fiber types with MHC I, IIa, IIx, and IIb isoforms.³⁰ (link)^,47 (link) The other serial cryosection underwent immunofluorescent staining with a primary antibody for GSK3β (9315, Cell Signaling, 1:2500 dilution) and an anti-rabbit Alexa Fluor 647 fluorescent secondary antibody (A27040, ThermoFisher Scientific, 1:5000 dilution). Once fiber types were identified with MHC isoform expression/fluorescence, GSK3β was quantified by converting the image to grayscale and quantifying the mean gray value with ImageJ. Within a single muscle, 10 of each fiber type were randomly analyzed.