title: A double fluorescence staining protocol to determine the cross-sectional area of myofibers using image analysis.
abstract: 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.
authors.affiliation: Muscle Biology Laboratory, and Department of Anatomy, University of Wisconsin, Madison, 53706, USA.
authors.forename: P E
authors.initials: PE
authors.lastname: Mozdziak
authors.forename: T A
authors.initials: TA
authors.lastname: Fassel
authors.forename: E
authors.initials: E
authors.lastname: Schultz
authors.forename: M L
authors.initials: ML
authors.lastname: Greaser
authors.forename: R G
authors.initials: RG
authors.lastname: Cassens
mesh_terms: 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
chemical_list: D005456:Fluorescent Dyes; D014966:Xanthenes; D003208:Concanavalin A; C034657:Texas red; D016650:Fluorescein-5-isothiocyanate