Key result
Epifluorescence microscopy reveals a positive correlation between immunofluorescent signal optical density and muscle section thickness.
Immunofluorescent signals in skeletal muscle cryosections can be reliably quantitated by epifluorescence microscopy.
Supports quantitative immunofluorescence in preclinical muscle research; leaves open clinical translation from this animal model.
Quantitative analysis of signal intensities in immunostained sections has been performed in only a few studies owing to difficulties with quantifying amounts of antigen present. We determined correlations between fluorescent signal intensities and amounts of antigen in muscle cryosections by altering section thickness from 4 to 10 microm. Fluorescent signals of dystrophin. beta-dystroglycan and myosin were detected with monoclonal and/or polyclonal primary antibodies using routine procedures. Confocal laser microscopy demonstrated that these signals were distributed uniformly along the z-axis suggesting that the antibodies permeated well through the sections. Epifluorescence microscopy with microfluorometry demonstrated a positive correlation between the optical density of signals and section thickness. These findings suggest that immunofluorescent signals can be quantitated by epifluorescence microscopy.
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Kawajiri et al. (1999) studied this question. Altering section thickness (4 to 10 microm) was evaluated on Correlation between optical density of signals and section thickness. Epifluorescence microscopy with microfluorometry demonstrated a positive correlation between the optical density of immunofluorescent signals and muscle section thickness.
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