Histologic and immunostaining analysis

For histological and immunostaining evaluation, all harvested tissue samples were fixed using 4% PFA overnight, followed by 1× PBS washing 3 times. Then the tissues were dehydrated using a 30% saccharose solution for 12 h. The samples were immersed in an optimal cutting temperature (OCT) compound and quickly frozen in liquid nitrogen. The samples were cryosectioned into 10-µm slides. H&E and Masson’s trichrome staining was performed according to the manufacturer’s instructions. Images were captured on a Leica DM1000 microscope.

For immunofluorescence, the slides were blocked using 5% Albumin BSA in PBS supplemented with 1% Triton X-100 for 1 h at room temperature. Then the sides were incubated at 4 °C for 24 h with rabbit anti-desmin (1:500, Servicebio, GB11081), rabbit anti-MYH7 (1:400, Servicebio, {"type":"entrez-nucleotide","attrs":{"text":"GB111857","term_id":"336599336"}}GB111857), mouse anti-human/mouse/rat/chicken Pax7 (5 µg/mL; R&D Systems, MAB1675), rabbit anti-CD31 (1:200, Servicebio, GB11063-3), rabbit anti-α-SMA (1:100, Servicebio, {"type":"entrez-nucleotide","attrs":{"text":"GB111364","term_id":"336598843"}}GB111364), rabbit anti-NF (1:100, Abcam, ab223343), rabbit anti-βIIIT (1:100, Abcam, ab229590), rat anti-CD68 (1:100, Invitrogen, Cat# 14-0681-82), rabbit anti-CD3 (1:100, Abcam, ab16669), rabbit anti-Ki67 (1:600, Servicebio, {"type":"entrez-nucleotide","attrs":{"text":"GB111141","term_id":"336598620"}}GB111141), rabbit anti-HLA (1:100, Abcam, ab52922). Slides were then washed by 1× PBS washing for 3 times and incubated in the secondary antibody such as anti-rabbit secondary antibody (1:400) (Alexa Fluor 488, Abcam), anti-rabbit secondary antibody (1:400) (Alexa Fluor 594, Abcam), anti-mouse secondary antibody (1:400) (Alexa Fluor 488, Abcam), anti-rat secondary antibody (1:400) (Alexa Fluor 488, Abcam) and anti-rat secondary antibody (1:400) (Alexa Fluor 568, Abcam) at 4 °C for 24 h. Finally, the slides were mounted using DAPI (1:5000) and the images were acquired under a confocal microscope (Nikon A1). Negative control was performed in parallel.