Quantitative analysis of immunofluorescent signals for dystrophin, beta-dystroglycan and myosin skeletal muscle by epifluorescence microscopy

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.