Di 8 anepps

Wild-type and MDX myofibers were stained with the voltage-sensitive dye pyridinium, 4-[2-(6-(dioctylamino)-2-naphthalenyl) ethenyl]-1-(3-sulfopropyl)-, inner salt (di-8-ANEPPS; Life Technologies, Carlsbad, CA, Cat. No. D3167). Myofibers were incubated with di-8-ANEPPS (2.5 μmol/L per L in DMEM media) for 3 h at 37°C, washed in L-15 media plus 2.5 μmol/L di-8-ANEPPS, and then imaged on a Fluoview 500 confocal system (Olympus; ×60, 1.3 NA water-immersion objective; pixel dimensions 0.2 × 0.2 μm in x and y) using L-15 media. Confocal images (512 × 512 pixels) of the tubular network were obtained from randomly selected myofibers using the same image acquisition settings and enhancing parameters. All malformed myofibers observed were used for imaging. Images were background corrected and a region of interest (ROI) of fixed dimensions was used to estimate the average fluorescence profile within the region of interest.

Myofibers were stained with the voltage-sensitive dye pyridinium, 4-[2-(6-(dioctylamino)-2-naphthalenyl) ethenyl]-1-(3-sulfopropyl)-, inner salt (di-8-ANEPPS; Thermo Fisher, Cat. No. D3167) as previously described [45 (link), 52 (link)]. Briefly, myofibers were incubated with di-8-ANEPPS 2.5 μM/L in MEM media for 2- 4 h at 37 °C and then imaged on a Fluoview 500 confocal system (Olympus, Waltham, MA); using a × 60, 1.3 NA water-immersion objective using L-15 media (ionic composition in mM: 137 NaCl, 5.7 KCl, 1.26 CaCl₂, 1.8 MgCl₂, pH 7.4; Thermo Fisher, Cat. No.21083-027) as recording solution. Confocal images (512 × 512 pixels) of the tubular network were obtained from randomly selected myofibers using the same image acquisition settings and enhancing parameters. Images were background corrected, and a region of interest of fixed dimensions was used to estimate average fluorescence profile within the region of interest.