Casted gel constructs or dissected muscles were washed in PBS and embedded in OCT compound and were frozen using precooled isopenthane. Embedded tissues were then sectioned at 8–12mmthickness and fixed in 4% paraformaldehyde, rehydrated and stained with Lac Z (β-Gal).
For immunostaining, the slides were washed with PBS, treated and permeablized with 0.03%Triton X-100 (Sigma, St. Louis, Mo), and blocked with 3% BSA (Amresco, Solon, OH) before applying the primary antibodies. Primary antibodies include rabbit anti-von Willebrand factor (Dako, Carpipenteria, CA), rabbit anti-laminin antibody (Sigma, St. Louis, MO), mouse anti-Pax7 antibody (R&D), mouse anti-Myf5 antibody (SCBT), mouse anti-myogenin (BD Pharmingen) and mouse antimyosin heavy chain-MHC (MYH1) antibody (MF20, DSHB). After incubation, the slides were all washed and stained with appropriate secondary antibodies (goat anti-rabbit Alexa Fluor 555 for the vWF stain, goat anti-mouse Alexa Fluor 555 for the PAX7, Myf5, myogenin and MHC and goat anti-rabbit Alexa Fluor 488 for the Laminin- Life technologies, Carlsbad, CA). The slides were then washed and mounted with Pro–long Gold anti-fade DAPI mounting media (Life Technologies, Grand Island, NY). For the evaluation of the muscle/fibrosis, slides were stained for Lac-Z, H&E or mason trichrome staining.
For immunostaining, the slides were washed with PBS, treated and permeablized with 0.03%Triton X-100 (Sigma, St. Louis, Mo), and blocked with 3% BSA (Amresco, Solon, OH) before applying the primary antibodies. Primary antibodies include rabbit anti-von Willebrand factor (Dako, Carpipenteria, CA), rabbit anti-laminin antibody (Sigma, St. Louis, MO), mouse anti-Pax7 antibody (R&D), mouse anti-Myf5 antibody (SCBT), mouse anti-myogenin (BD Pharmingen) and mouse antimyosin heavy chain-MHC (MYH1) antibody (MF20, DSHB). After incubation, the slides were all washed and stained with appropriate secondary antibodies (goat anti-rabbit Alexa Fluor 555 for the vWF stain, goat anti-mouse Alexa Fluor 555 for the PAX7, Myf5, myogenin and MHC and goat anti-rabbit Alexa Fluor 488 for the Laminin- Life technologies, Carlsbad, CA). The slides were then washed and mounted with Pro–long Gold anti-fade DAPI mounting media (Life Technologies, Grand Island, NY). For the evaluation of the muscle/fibrosis, slides were stained for Lac-Z, H&E or mason trichrome staining.