TA muscles from WT and BAI3 KO mice were dissected and embedded with OCT compound. To analyze the number of myonuclei per fibers, 10 μm frozen sections were fixed in 4% paraformaldehyd and permeabilized with a PBS/0.2% Triton X-100 solution for 10 min. Sections were incubated in blocking buffer (PBS/1% BSA) for 1 h. Cells were next incubated with anti-laminin DyLight 650 (dilution 1:250; Novus) and Hoechst (dilution 1:10,000; Invitrogen).
To analyze the number of Pax7-positive cells, 10 μm frozen sections were fixed in 4% paraformaldehyde for 10 min. Slides were boiled for 20 min in antigen retrieval buffer (10 mM Sodium Citrate pH 6.0) prior to incubation in blocking buffer (10% Goat serum/0.4% TritonX/PBS) for 1 h. Muscle sections were incubated with primary antibody Pax7 (dilution 1:10 (Developmental Hybridoma)) diluted in 0.04% TritonX/1%BSA/PBS at 4 °C overnight. Following three washes of PBS, sections were incubated with secondary antibody Alexa 568 conjugated goat anti-mouse secondary antibody (dilution 1:300; Invitrogen) and anti-laminin DyLight 650 (dilution 1:250; Novus) for 1 h. Hoechst (dilution 1:10,000; Invitrogen) was used to reveal nuclei. Pictures were taken with the DM6 microscope (Leica) at an objective of ×20 and the images were analyzed using the Volocity software.
To analyze the number of Pax7-positive cells, 10 μm frozen sections were fixed in 4% paraformaldehyde for 10 min. Slides were boiled for 20 min in antigen retrieval buffer (10 mM Sodium Citrate pH 6.0) prior to incubation in blocking buffer (10% Goat serum/0.4% TritonX/PBS) for 1 h. Muscle sections were incubated with primary antibody Pax7 (dilution 1:10 (Developmental Hybridoma)) diluted in 0.04% TritonX/1%BSA/PBS at 4 °C overnight. Following three washes of PBS, sections were incubated with secondary antibody Alexa 568 conjugated goat anti-mouse secondary antibody (dilution 1:300; Invitrogen) and anti-laminin DyLight 650 (dilution 1:250; Novus) for 1 h. Hoechst (dilution 1:10,000; Invitrogen) was used to reveal nuclei. Pictures were taken with the DM6 microscope (Leica) at an objective of ×20 and the images were analyzed using the Volocity software.