Goat anti mouse igg2b af488

Muscle sections were fixed in 4% PFA for 10 minutes, washed in PBS, then incubated in 0.5% Triton X-100 diluted in PBS for 10 minutes. Sections were then rinsed in PBS and blocked in 2.5% normal horse serum (NHS) (S-2012; Vector Labs) for 90 minutes. Following blocking, sections were incubated in primary antibodies overnight for rabbit IgG anti-p16 (1:250, ab108349; Abcam) and mouse IgG2b anti-dystrophin (1:200, D8168 Sigma; Millipore Sigma) diluted in NHS. For p16, two additional antibodies were used (A0262, ABclonal; MAB4133, Millipore Sigma), however, they did not result in any positive staining. The following morning, sections were rinsed in PBS and then incubated in goat anti-rabbit IgG biotin (1:1000; Jackson ImmunoResearch) diluted in NHS for 75 minutes. Sections were then rinse in PBS and incubated in streptavidin-conjugated AF594 (1:250; Invitrogen) and goat anti-mouse IgG2b AF488 (1:250, A21141; Invitrogen) diluted in PBS for 75 minutes. Sections were then rinsed in PBS, stained with DAPI (1:10,000; Invitrogen) for 10 minutes and mounted with VectaShield fluorescent mounting media (Vector Labs).