Alexa 568 conjugated goat anti mouse secondary antibody

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.

C2C12 and Sol8 myoblasts were fixed in 4% paraformaldehyde and permeabilized with a PBS/0.2% Triton X-100 solution for 10 min prior to incubation in blocking buffer (PBS/1% BSA) for 1 h. Cells were next incubated with the primary antibody recognizing MyHC (MF20, dilution 1:20 (Developmental Hybridoma)) diluted in blocking buffer. Cells were washed with PBS and incubated with an Alexa 568 conjugated goat anti-mouse secondary antibody (dilution 1:2500; Invitrogen) for 1 h. Hoechst (dilution 1:10,000; Invitrogen) was used to reveal nuclei. GFP/mVenus positive chick embryos were fixed in 4% paraformaldehyde for 1 h at room temperature and washed three times with PBS. They were then incubated overnight in 30% sucrose solution at 4 °C and embedded in OCT prior to cryosectioning. 8 µm sections were stained as described above for cells with the following modifications: sections were washed in PBS/0.1% Triton X-100 and the Alexa 568 conjugated goat anti-mouse secondary antibody was used at a 1:2500 dilution. Pictures were taken with the Axiovert microscope (Zeiss) at an objective of ×20 and with a confocal microscope LSM700 (Zeiss) at an objective of × 100. In both cases, the images were analyzed using the Volocity software, as described above.