Goat anti mouse or goat anti rabbit igg alexa fluor 488 antibody

FACS-isolated MuSCs from age and sex-matched animals were fixed in 1.6% PFA/PBS for 10’ on ice, spun at 350xg at 4°C, and stored in cold 100% methanol (final concentration 90%) at −80°C until the staining commenced. MuSCs were further permeabilized in 1% Igepal CA-630/PBS, washed with PBS, and blocked with 1% BSA/PBS/0.1% Triton X-100, and stained with phospho-p65 antibody (Abcam, 1/500), or phospho-JNK (1/100, Cell Signaling), phospho-p38 (1/100, BD Biosciences), phospho-S6K (1/100, Cell Signaling), phospho-AKT (1/100, Cell Signaling), or Bcl2 (1/100, BD Biosciences) in 1%BSA/PBS/0.1% Triton X-100 overnight at 4°C in V-bottom 96-well plates. MuSCs were washed in BSA buffer, stained with goat anti-mouse or goat anti-rabbit IgG Alexa Fluor 488 antibody (Thermo, 1/500), and analyzed on an LSRII flow cytometer using the 488nm laser line 530/30 bandpass filter set. Data were analyzed with FlowJo. Unbiased analysis was performed with the investigators blinded to genotypes and/or conditions. Results were consistent and reproducible with different batches of Abs, using MuSC isolation from different experimental mice performed on different days. Antibody specificity was determined by several methods, including using unstained controls, no primary antibody-stained controls, and FMO controls.