A double fluorescence staining protocol to determine the cross-sectional area of myofibers using image analysis.
A double fluorescence staining protocol was developed to facilitate computer based image analysis. Myofibers from experimentally treated (irradiated) and control growing turkey skeletal muscle were labeled with the anti-myosin antibody MF-20 and detected using fluorescein-5-isothiocyanate (FITC). Extracellular material was stained with concanavalin A (ConA)-Texas red. The cross-sectional area of the myofibers was determined by calculating the number of pixels (0.83 mu m(2)) overlying each myofiber after subtracting the ConA-Texas red image from the MF-20-FITC image for each region of interest. As expected, myofibers in the irradiated muscle were smaller (P < 0.05) than those in the non-irradiated muscle. This double fluorescence staining protocol combined with image analysis is accurate and less labor-intensive than classical procedures for determining the cross-sectional area of myofibers.
Mozdziak
P E
PE
Muscle Biology Laboratory, and Department of Anatomy, University of Wisconsin, Madison, 53706, USA.
Fassel
T A
TA
Schultz
E
E
Greaser
M L
ML
Cassens
R G
RG
D000818:Animals; D003208:Concanavalin A; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D007091:Image Processing, Computer-Assisted; D008297:Male; D008867:Microtomy; D009210:Myofibrils / Q000528:radiation effects / Q000648:ultrastructure*; D013194:Staining and Labeling / Q000379:methods*; D014422:Turkeys; D014966:Xanthenes
D005456:Fluorescent Dyes; D014966:Xanthenes; D003208:Concanavalin A; C034657:Texas red; D016650:Fluorescein-5-isothiocyanate