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

Quantitative analysis of signal intensities in immunostained sections has b een 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 alterin g section thickness from 4 to 10 mu m, Fluorescent signals of dystrophin, b eta-dystroglycan and myosin were detected with monoclonal and/or polyclonal primary antibodies using routine procedures. Confocal laser microscopy dem onstrated that these signals were distributed uniformly along the z-axis su ggesting that the antibodies permeated well through the sections. Epifluore scence microscopy with microfluorometry demonstrated a positive correlation between the optical density of signals and section thickness, These findin gs suggest that immunofluorescent signals can be quantitated by epifluoresc ence microscopy.