Ab52631

Paraffin-embedded ankle tissue was sectioned and rinsed with PBS after dewaxing. After deparaffinization and the removal of catalase by adding 3% H₂O₂, the antigen sites were exposed by steaming twice after washing. After antigen retrieval, blocking was performed with serum. After blocking was completed, MMP-1 (Abcam, # ab52631) and MMP-3(Abcam, # ab52915) antibodies were added at 4°C overnight. After removal, the tissue was washed in PBS, followed by incubation with the secondary antibody. After washing in PBS, the color reagent was added. Finally, the specimens were observed and photographed under a microscope [14 ].

Paraffin-embedded ACLs were sectioned longitudinally (5 μm thick). In order to perform the staining procedure, first these sections were deparaffinized, rehydrated, and then antigen retrieval was accomplished by 10 mM citrate buffer at 60 °C for 1 h. These sections were incubated with rabbit primary antibodies against MMP-1 (1:100), MMP-3 (1:50), or IL-6 (1:400) at 4 °C overnight, rinsed with phosphate-buffered saline, and incubated with reagents included in the Histofine SAB-PO(R) kit (Nichirei Biosciences Inc., Tokyo, Japan). All sections were visualized using the Olympus BX53 microscope (Olympus, Tokyo, Japan); images were captured digitally using the Leica DFC7000 T digital camera and processed using the Leica Application Suite Ver4.6 imaging software. The primary antibodies used were rabbit anti-MMP1 antibody (1:100, ab52631, lot: GR3261996-3; Abcam, Cambridge, UK), rabbit anti-MMP3 antibody (1:50, ab52915, lot: GR3249690-2; Abcam), and rabbit anti-IL-6 antibody (1:400, ab6672, lot: GR3195128-25; Abcam). The acquired images were analyzed using ImageJ Fiji (https://imagej.net/software/fiji/). Color deconvolution in ImageJ Fiji was used to separate the 3,3′-diaminobenzidine staining, and the mean gray values in the nucleus and cytoplasm were measured and quantified.

Using a Cell Mitochondria Isolation Kit (Beyotime, Shanghai, China), we extracted proteins from target cells. The samples were incubated with the following primary antibodies: anti-TGFβ1 (ab21610, Abcam, Cambridge, MA, USA), anti-TGFβ1 (ab64715, Abcam), anti-α-SMA (ab5694, Abcam), anti-p-SMAD2 (ab53100, Abcam), anti-SMAD2 (ab40855, Abcam), anti-MMP1 (ab52631, Abcam), anti-MMP3 (ab52915, Abcam), anti-ACTG2 (SAB1411364, Sigma-Aldrich, St. Louis, MO, USA), and anti-GAPDH (ab8245, Abcam) overnight at 4°C; afterward, the samples were incubated with goat anti-rabbit IgG polyclonal antibody (Abcam) or goat anti-mouse IgG polyclonal antibody (Abcam) for 1 h at room temperature. Visualization was conducted using an enhanced chemiluminescence (ECL) detection system (Thermo Fisher Scientific) using GAPDH levels as an endogenous control.

We extracted the total protein by RIPA lysate. Then, we separated 30 μg protein on 10% protein electrophoresis gel and then transferred it to the PVDF membrane (Invitrogen). The PVDF membrane was blocked with 5% skim milk powder for 1-2 h, and then anti-collagen III (COLIII) (1 : 1000, Abcam, ab184993), anti-α smooth muscle actin (α-SMA) (1 : 500, Abcam, ab5694), anti- matrix metalloproteinase (MMP)-1 (1 : 1000, Abcam, ab52631), anti-MMP-8 (1 : 1000, Abcam, ab81286), anti-NFAT5 (1 : 1000, Abcam, ab3446), antitumor necrosis factor-α (TNF-α) (1 : 1000, Abcam, MA5-23720), interleukin (IL)-6 (1 : 500, Abcam, M620), anti-IL-1β (1 : 500, Abcam, M421B), and anti-β-actin (1 : 1000, Abcam, ab6276) were added and incubated overnight 4 °C. The next day, the PVDF membranes were incubated in HRP anti-mouse IgG for IP (1: 1000, Abcam, ab131368) at 25 °C for an h. The bands were detected using an exposure meter (Bio-Rad, USA) with hypersensitive chemiluminescence (HRP) detection kit (LMAI Bio, Shanghai, China).

The tissues were fixed in 10% neutral-buffered formalin (BBC Biochemical, Mount Vernon, WA, USA) and embedded within paraffin. The paraffin-embedded samples were sliced into 5 μm sections and histological observation was performed following hematoxylin and eosin (H&E) staining. For immunohistochemistry, tissue sections were stained with an MMP-1 antibody (ab52631, Abcam, Cambridge, MA, USA) at 4 °C overnight. The expression level of MMP-1 in the dermis was quantified by using Image-Pro Plus 7 software (Media Cybernetics, Inc. Rockville, MD, USA).