Anti collagen 3

Heart sections were immersed in 0.01 M sodium citrate for antigen retrieval and treated with 3% H₂O₂ for 10 min to remove endogenous peroxidase. The sections were then incubated with 10% normal horse serum (NHS) for 1 h, followed by the primary antibodies anti-PUMA (1:200 dilution, Cell Signaling), anti-ETS-1 (1:200 dilution, Abcam), anti-fibronectin (1:50 dilution, Abcam), and anti-collagen 3 (1:50 dilution, Proteintech) overnight at 4 °C. Then, a second antibody specific to the primary antibody was selected and allowed to react at RT for 90 min. The avidin-biotin-peroxidase-complex was incubated for 1 h. Finally, the tissues were stained with DAB (Sigma) color reaction solution and observed with a light microscope. Additionally, after each designated treatment, the H9c2 cells on the coverslip were fixed in 4% paraformaldehyde for 15 min and then permeabilized with 0.1% Triton X-100 for 1 min at RT. The cells were washed again and then blocked for 1 h with 1% bovine serum albumin (BSA, Novagen) in PBS at RT. The cells were incubated with the previously identified antibodies. After being washed 3 times with PBS, the cells were incubated with anti-rabbit 488 (Invitrogen, A27034) at RT for 1 h. The nuclei were counterstained with DAPI for 5 min, and then the cells were observed and photographed under a fluorescence microscope.

The treated PMFs were lysed with radioimmunoprecipitation assay buffer (Beyotime) containing the protease inhibitor phenylmethylsulfonyl fluoride (Beyotime). The protein concentration was measured according to the instructions of a bicinchoninic acid protein assay kit (Beyotime). Equal volumes of protein samples were loaded onto a 10% SDS‐PAGE gel (Beyotime), electrophoretically separated, and transferred to polyvinylidene difluoride membranes (Millipore, MA). After blocking with 5% skim milk at room temperature for 2 hours, the membranes were incubated overnight with the following primary antibodies at 4 °C: anti–β‐actin (1:3000; Servicebio), anti–α‐SMA (1:1000; Servicebio), anti–collagen‐1 (1:1000; Servicebio), anti–collagen‐3 (1:1000; Proteintech), anti‐Shh (1:1000; Proteintech), anti‐Smoothened (1:1000; Affinity), and anti–Gli‐1 (1:1000; Novus). Then, the membranes were incubated with the following secondary antibodies for 1 hour at room temperature: HRP‐conjugated anti‐mouse secondary antibody (1:5000; Servicebio) and HRP‐conjugated anti‐rabbit secondary antibody (1:5000; Servicebio). The protein bands were visualized using HRP‐substrate chemiluminescence technology (Servicebio). For quantification, the intensity of each target protein band was normalized to that of the β‐actin band.

Briefly, paraffin-embedded rat skin was routinely de-paraffinized and re-hydrated. The samples were incubated with diluted primary antibodies at 4 °C overnight and then processed with the iVision™ Poly-HRP goat anti-mouse/rabbit secondary antibody reagent (Talentbiomedical, Xiamen, China, DD13) and ABC complexes. Finally, samples were reacted with a DAB kit (Servicebio, Wuhan, China, G1212-200T). Images were acquired with an optical microscope (CKX41, Olympus, Tokyo, Japan) and analyzed for IOD in each view.
The primary antibodies used were as follows:
Anti-Collagen III (Proteintech, Rosemont, IL, USA, 22734-1-AP, 1:500);
Anti-Fibronectin (Proteintech, Rosemont, IL, USA, 15613-1-AP, 1:500).