Goat anti rabbit 546 a11010

Primary antibodies were used at 0.5–1 μg ml⁻¹ and sourced from Abcam: rabbit anti-Ki67 (ab16667, 1:200), and rabbit anti-pSmad3 (1:200); Fisher Scientific: rabbit anti-follistatin (PA519787, 1:200); Santa Cruz Biotechnologies: anti-Sox2 (sc-17320 1:400); R&D Systems mouse anti-Albumin (MAB1455, 1:1000), rabbit anti-myostatin (AF788, 1:200). Mouse hybridoma anti-eMHC was prepared in house (F1.652 clone, Developmental Studies Hybridoma Bank, University of Iowa, deposited by Blau, HM) and used at 1:50., Secondary fluorochrome conjugated antibodies were from Life Technologies: goat anti-rabbit 546 (A11010, 1:2,000) and goat anti-mouse 488 (A11029, 1:2,000). DNA was stained by Hoechst 33342 from Sigma Aldrich (B2261) used at 1 μg ml⁻¹. Negative controls with isotype-matched immunoglobulin G (IgGs) were routinely performed for all immunodetection studies shown here and above; and background non-specific immunofluorescence was minimal, Supplementary Fig. 8.

Muscle sections attached to positively charged frosted glass microscope slides were fixed with 70% ethanol at 4 °C overnight and rinsed with 1× phosphate-buffered saline (PBS) the next day. The sections were then blocked for 30 min in 1% staining buffer (1% calf serum in 1× PBS). Samples were permeabilized with 0.1% Triton X-100 in 1X PBS for 5 min at room temperature and subsequently rinsed with 1% staining buffer 3 times, 5 min per rinse. The sections were incubated with primary antibodies diluted to 0.5–1 μg/ml at 4 °C overnight. The slides were rinsed as stated and then coated with secondary antibodies and Hoechst nuclear DNA stain for 2 h at room temperature in the dark. Samples were rinsed with PBS and mounted with Fluoromount. A Zeiss Axioscope fluorescent microscope was used for imaging. antibodies: rabbit anti-GFP antibody (abcam290,1:5000), rabbit anti-Ki67(ab16667, 1:200), and mouse anti-dystrophin antibody (Santa Cruz Biotechnology 58754,1:200) were used at dilutions according to manufacturer’s instructions. Secondary fluorochrome conjugated antibodies were from Life Technologies: goat anti-rabbit 546 (A11010, 1:2000) and goat anti-mouse 488 (A11029, 1:2000). DNA was stained by Hoechst 33342 from Sigma Aldrich (B2261) 1:500 at 1 μg/mL.