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.
Kawajiri
M
M
First Department of Internal Medicine, School of Medicine, University of Tokushima, Japan.
Mitsui
T
T
Kawai
H
H
Shono
M
M
Matsumoto
T
T
D000328:Adult; D003598:Cytoskeletal Proteins / Q000032:analysis*; D049030:Dystroglycans; D016189:Dystrophin / Q000032:analysis*; D006801:Humans; D008562:Membrane Glycoproteins / Q000032:analysis*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000473:pathology; D009218:Myosins / Q000032:analysis*
D003598:Cytoskeletal Proteins; C483081:DAG1 protein, human; D016189:Dystrophin; D008562:Membrane Glycoproteins; D049030:Dystroglycans; D009218:Myosins