Horseradish peroxidase conjugated avidin biotin complex

After fixation and dehydration in 30% sucrose phosphate buffer, parts of each animal's back muscles were embedded with embedding medium (OCT, SAKURA, USA) and cut at 20 μm thickness frozen section with a microtome. Then, the sections were incubated successively with primary antibodies, Anti-MYOD antibody (Abcam, 1 : 1000), Anti-p38 MAPK (sc-1661821 : 1000), the secondary antibody, goat biotinylated conjugated polyclonal anti-rabbit antibody (1 : 250; Vector Laboratories), and horseradish-peroxidase-conjugated avidin-biotin complex (Vector Laboratories). Sections were then exposed to diaminobenzidine (DAB) for detection. To adequately quantify MYOD and p38 MAPK positive cells, we used the nuclear counterstain methyl green (Vector Laboratories) and counted MYOD and p38 MAPK positive cells.

Lung sections (4 μm thick) were de-paraffinized with 3 xylene washes and re-hydrated by successive washes in 100, 90, and 70% ethanol, and then finally in dH₂O. Heat-based antigen retrieval was performed by heating slides in citrate buffer (pH 6.0) to 120°C in a pressure cooker. Slides were then incubated with 5% donkey serum for 30 min and endogenous peroxidase was inhibited with 0.3% H₂O₂ treatment for 20 min. Slides were incubated with the primary antibody overnight, the biotinylated secondary antibody for 30 min, and horseradish peroxidase-conjugated avidin-biotin complex (Vector Laboratories, Burlingame, CA, United States) for 45 min. Staining was visualized with 3-amino-9-ethyl carbazole (AEC) substrate chromagen, which results in red staining (Vector Laboratories). Sections were then counterstained with hematoxylin. The following primary antibodies were used: Mac-3 (5 μg/mL) (BD Biosciences, Franklin Lakes, NJ, United States) to visualize macrophages and myeloperoxidase (MPO) to visualize neutrophils (5 μg/mL) (Abcam, Cambridge, MA, United States). Immune cells were manually counted on a microscope. Quantification of the staining was performed in a manner that was blinded from knowledge of specific mouse IDs. The total lung area for each section was quantified in ImageJ and Mac-3-positive or MPO-positive cells were expressed per total area (mm²).