Alexa fluor 568 goat anti mouse igg2b

Histological cross sections (8 μm) were collected from skeletal muscles using a cryostat and maintained in blocking solution (10% goat serum diluted in PBS) for 30 minutes in a humid chamber. Slides were stained overnight at 4°C with PBS containing either or a combination of anti-Myh7 (BA-F8, 1:50, Developmental Studies Hybridoma Bank [DSHB]) primary antibody, anti-Myh2 primary antibody (SC-71, 1:20, DSHB), anti-Myh4 primary antibody (BF-F3, 1:10, DSHB) and with anti-laminin antibody (1:200, L9393, MilliporeSigma) to delineate myofiber outlines present in a muscle section. Immunoglobulin M (IgM) primary antibody conjugated to FITC (1:300, SAB4700348, MilliporeSigma) was used to identify myofibers with compromised membrane integrity. Primary antibodies were visualized using Alexa Fluor 568 goat anti-mouse IgG2b (Invitrogen), Alexa Fluor 488 goat anti-mouse IgG2b (Invitrogen), Alexa Fluor 674 anti-mouse IgG1, Alexa Fluor 568 IgGm, and Alexa Fluor 405 or 568 goat anti-rabbit IgG secondary antibodies diluted 1:500 in PBS. Immunofluorescence images were captured using a Nikon Eclipse Ti microscope. Entire muscle sections were analyzed for type I positive fibers. All experiments were performed in a blinded fashion whereby the experimenter was only made aware of the genotypes after the quantification was performed.