Quantifying Muscle Fiber Morphology

To capture single images or z-series stacks, we used an AxioImager microscope (Zeiss), filters for DAPI (excitation/emission 365/445; Zeiss filter set 49), GFP (excitation/emission 470/525; Zeiss filter set 38), Cy3 (excitation/emission 550/605; Zeiss filter set 43 HE), and Cy5 (excitation/emission640/690; Zeiss filter set 50), a Flash 4.0 LT sCMOS camera (Hamamatsu), and Volocity image acquisition software (version 6.3.1; Perkin Elmer). Objectives were ×5 EC Plan-NEOFLUAR NA 0.16, ×10 EC Plan-NEOFLUAR NA 0.3, ×20 Plan-APOCHROMAT NA 0.8, ×40 Plan-APOCHROMAT NA 1.4, and ×63 Plan-APOCHROMAT NA 1.4. To quantitate fluorescence, we used Volocity quantitation and restoration software modules (version 6.3.1; Perkin Elmer), as previously described^{30 (link)}. We measured minimal Feret’s diameter, defined as the minimum distance between parallel tangents^{37 (link)}, by MyHC fiber type in 8 µm frozen sections of TA muscle fibers of LR41;Mbnl1^−/− mice treated with the Clcn1 ASO or invert oligo. Untreated WT served as controls.