Masson s trichrome staining

For immunofluorescence staining, hearts were fixed with 4% paraformaldehyde, dehydrated in 30% sucrose, and then cut into 5 μm sections. The fixed cultured cells and heart tissue sections were blocked with 30% normal goat serum for 40 min, incubated with primary antibodies overnight at 4°C, and subsequently incubated with secondary antibodies (1:500 dilution) in the dark at room temperature for 1 h. The following primary antibodies were used: anti-CD51 (1:100, Abcam, United Kingdom), Sca-1 (1:50, Santa Cruz, United States), C-kit (R&D, United States), anti-CD31 (1:200, Abcam), and anti-Ki67 (1:200, Abcam). For immunohistochemistry, hearts were fixed overnight with 10% formalin and then embedded in paraffin. The processed paraffin slides (5 μm) were blocked with normal goat serum, incubated with a monoclonal anti-CD31 antibody (1:200, Abcam) overnight at 4°C, developed in DAB, and counterstained with hematoxylin according to the manufacturer’s instructions. Endothelialization was calculated as the ratio of the surface covered by the number of vessels stained by CD31. To evaluate the infarct size, paraffin sections of hearts (4 weeks post MI) were subjected to Masson’s trichrome staining (Servicebio, China).

Paraformaldehyde (4%) fixed hearts were sectioned transversely at the mid-ventricular level, embedded in paraffin. Five-μm-thick sections were generated, stained with Masson's trichrome staining (Servicebio, Wuhan, China) according to the manufacturer's instructions 90 (link), and then examined by light microscopy. For each slide, the percentage of fibrotic area was quantified by manually tracing the blue area in five randomly selected microscopic fields using Image-Pro Plus software 6.0 (Media Cybernetics, Bethesda, MD, USA) 90 (link). The investigator performing fibrotic area analysis was blinded to group allocation.

Masson’s trichrome staining (G1006, Servicebio, China) was performed in kidney paraffin sections following the manufacturer’s instructions.

The specimens of kidney and colon tissue stored in 4% neutral buffered formalin were embedded in paraffin after routine histological processing. Then, these paraffin-embedded specimens were cut into sections at thickness of 5 μm. After routinely dewaxing and hydration, the sections were dyed with haematoxylin and eosin (H&E, Servicebio, Wuhan, China) and Masson's trichrome staining (Servicebio, Wuhan, China). Histology was performed at magnification of ×400, and pathology and morphological analyses were independently performed by an experienced pathologist blinded to the protocol.

Frozen sections or paraffin sections in groups were prepared as described above. Re-epithelialization and scar formation were assessed by H&E staining (Servicebio, China). Collagen deposition was detected by Masson’s trichrome staining (Servicebio, China). For immunohistochemical staining, the sections were blocked with 1% bovine serum albumin (Servicebio, China) and 0.5% Triton-X100 (Servicebio, China) and then separately treated with AE1/AE3 (Abcam, USA, prediluted), CD31(Abcam, USA, 1:500), CD68 (Abcam, USA, 1:500) and P63 (Abcam, USA, 1:500) mouse anti-human IgG antibody at 4 °C overnight. After washing three times with PBS for 5 min, the sections were incubated for 1 h with an HRP goat anti-mouse IgG antibody (Invitrogen, USA, 1:1000) at room temperature. Finally, the sections were stained with 3,3 N-diaminobenzidine tetrahydrochloride and counterstained with hematoxylin. The slides were covered with coverslips and examined under a microscope.

The acquired sample segments were submerged in 10% paraformaldehyde for 24 h. A paraffin microtome (RM2235, Leica, Wetazlar, GER) was used to slice samples embedded in paraffin blocks into 5-m thick sections.The slides were rehydrated dried and given a PBS washing. The slides were subsequently stained with hematoxylin and eosin (HE) (Servicebio, Wuhan, China) for histological analysis to gauge the percentage of inflammatory cells. Masson’s trichrome staining (Servicebio) was used to examine the density of collagen fibers and variations in collagen under a microscope.Subsequently, the slides were then treated with rabbit anti-ASPN antibody, rabbit anti-SMA antibody, and rabbit anti-collagen I antibody overnight at 4°C each and incubated with the secondary antibody at room temperature for 2 h. The slides were next stained with Meyer’s hematoxylin and 3,3-diaminobenzidine (DAB; Sigma, St. Louis, Missouri, United States) for 1–2 min (Sorabio, Beijing, P.R.C.). After blocking with neutral gum (Invitrogen, San Diego, CA, United States), the slides were examined under a microscope (Leica DMR 3000; Leica, Bensheim, Germany). ImageJ was used to quantitatively analyse the immunohistochemistry image data.