Dab solution

Tumor tissue sections (5 μM) were made and used for immunohistochemical analysis. After paraffin removal, antigen repair, and removal of peroxidase from the sections, sections were incubated with indicated primary antibodies. Then slides were stained using DAB solution (diaminobenzedine, MXB Biotechnologies, Fuzhou, China), then counterstained with haematoxylin. Slices were photographed with Image Analyse system of Leica Microscope. Four low-power views (200×) were randomly selected from each slice.

TA muscle sections were fixed with 4% PFA and permeabilized with 0.2% Triton X-100 for 10 min in PBS. Then, sections were incubated with 3% H₂O₂ for 20 min to remove the endogenous peroxidase and blocked with 10% normal goat serum for 2 h at room temperature. Sections were incubated with primary Ab against eMyHC (F1.652, Developmental Studies Hybridoma Bank, Iowa City, IA, USA; 1:50), laminin (L9393, Sigma-Aldrich; 1:100), Pax7 (AF7584, Affinity Biosciences; 1:300), F4/80 (DF2789, Affinity Biosciences; 1:500) or MyoG (67082-1-Ig, Proteintech; 1:300) at 4°C overnight. For immunofluorescence, sections were treated with FITC- or TRITC-conjugated secondary Abs for 2 h and DAPI for 10 min to observe the muscle with a confocal microscope (Nikon). For immunohistochemistry, sections were incubated with biotinylated goat secondary Ab specific to the host species at room temperature for 1 h, and then visualized by DAB solution (MXB Biotechnologies, Fuzhou, China) for 5 min. The images were captured using a phase-contrast microscope.