Ab240654

We followed the methods of Sun et al. [17 (link)]. Four percent paraformaldehyde-fixed mouse lung tissues were cut into 4 μm thick sections. After dehydration, the sections were collected on Superfrost Plus glass slides. Sections were rinsed with PBS and permeabilized with a 1% Triton solution for 5 min. The cells were then blocked with 1% BSA for 1 h. Primary antibodies against α-SMA (Abcam, Ab240654, 1:200), SENP1 (Abcam, Ab236094, 1:200), and Sca-1 (Abcam, Ab51317, 1:200) were incubated with the sections overnight at 4 °C. Secondary antibodies were incubated with the samples for 1 h at room temperature. The sections were mounted with Fluorescent Mounting Media with DAPI and observed under a Leica SP8 confocal laser scanning microscope at magnifications of × 200.

Mice were sacrificed in different groups on Days 1, 3, 7, 11 and 14, and skin and skeletal muscle were harvested from within 1 cm² of the injury site. Tissues were fixed in 4% paraformaldehyde, paraffin-embedded and sectioned. The sections were cut at a thickness of 5 μm. As described above, H&E and Prussian blue staining were performed. Pathologic changes of inflammatory cell infiltration and iron deposition were observed by light microscopy (×40 and ×200). Five selected fields of each section were quantitatively analyzed using ImageJ software.
In addition, the paraffin-embedded sections were deparaffinized and dehydrated in a graded series of alcohols. Sections were embedded in 0.5% Triton X-100 for 10 min. Sections were incubated with 5% goat serum for 30 min, followed by overnight incubation at 4°C with various primary antibodies CD206 (Abcam, no. ab64693), CD86 (Abcam, no. ab239075), CD31 (Abcam, no. ab222783) and a-SMA (Abcam, no. ab240654) (1:100). Slides were washed three times with PBS for 5 min each and sealed with a fluorescence-quenching sealer. Images were observed with inverted fluorescence microscopy. Five images from each group were randomly selected for analysis. The fluorescence’s integrated gray value was calculated using the Image Pro Plus 9.0 image analysis software.

We followed the methods of Sun et al. [17 (link)]. Four percent paraformaldehyde‐fixed, paraffin‐embedded blocks of lung tissues were cut into 4 μm thick sections. The sections were placed on polylysine-coated slides and incubated in a 60 °C oven. The slides were dewaxed using xylene and rehydrated using a gradient of alcohol concentrations. The slides were then placed in a microwave oven until the antigen retrieval solution reached 100 °C for 10 min one and for 2 min four times, cooled to room temperature for 20 min and washed with PBS for 5 min. Primary antibodies against CD90 (Cell Signaling, #13,801, 1:200; Abcam, Ab181469, 1:200), SENP1 (Abcam, Ab236094, 1:200), and alpha-smooth muscle actin (α-SMA) (Abcam, Ab240654, 1:200) were incubated with the slides overnight at 4 °C. Secondary antibodies were incubated with the samples for 1 h at room temperature. Then, the sections were mounted using Fluorescent Mounting Media containing 4′,6-diamidino-2-phenylindole (DAPI) (Abcam). Each tissue section was observed under a confocal laser scanning microscope (Leica SP8, Wetzlar, Germany) at magnifications of 200× and 400×, if necessary.

LR-MSCs were fixed with freshly prepared 4% paraformaldehyde for 10 min at room temperature. The cells were then washed three times with PBS. Cells under coverslips were then incubated in 1% bovine serum albumin (BSA). Primary antibodies against α-SMA (Abcam, Ab240654, 1:200), Collagen I (Abcam, Ab260043, 1:200; Abcam, Ab88147, 1:200), and SENP1 (Abcam, Ab236094, 1:200) were incubated with the cells overnight at 4 °C. After washing with PBS, secondary antibodies were incubated with the cells for 1 h at room temperature in a darkened humidified chamber. Finally, cells under coverslips were washed with PBS and mounted in fluorescent mounting medium with DAPI. Images were acquired using a Leica SP8 confocal laser scanning microscope at magnifications of × 200.

Each aortic valve specimen used in histopathology analyses was divided into three parts: fibrotic areas, calcific areas and ILH areas. The degree of calcification in the above areas increased in sequence, as previously described [34 (link)]. All parts of the specimens were fixed in 4% paraformaldehyde and decalcified with 10% EDTA. After the tissues were embedded in paraffin, 4 μm sections were prepared using a Leica microtome.
The aortic valve sections were stained according to the instructions of four-color multiplex fluorescence immunohistochemical staining kit (Absin) and blocked with TBST containing 5% goat serum before incubation with antibodies. The antibodies involved in experiments include MRC1 (CST, 24,595 S), α-SMA (abcam, ab240654) and BMP2 (abcam, ab214821). The nuclei were stained with DAPI before sealing, and all sections were scanned by a fluorescent scanning camera (KFBIO, KF-TB-400). Inset images were captured by a confocal microscope. The areas of tissue in sections were calculated using image J according to the scale bars and numbers of positive cells were counted manually. Then the results were quantified as cell densities (cell numbers / tissue areas).
To perform z-stack scans of aortic valve tissue, 20 μm sections were prepared. Images were captured using a confocal microscopy (Zeiss LSM 710). 3D reconstruction was built in Zen2012 software (Zeiss).

The murine fibroblast line L-929 were purchased from Xiehe Hospital. The antibodies used in this study were as follows: anti-α-SMA (1:1000 for western blotting, 1:50,000 for immunohistochemistry [IHC], 1:50 for immunofluorescence [IF] ab240654, Abcam, UK), anti-COL1A1 (1:1000 for western blotting, 1:100 for IHC, 1:2,000 for IF, ab270993, Abcam, UK), anti-mouse fibronectin (FN) (1:1000 for western blotting, ab268020, 1:50 for IF Abcam, UK), anti-NOX4 (1:2000 for western blotting, 1:500 for IHC, 14347-1-AP, Proteintech, USA), anti-NLRP3 (1:2000 for western blotting, 1:200 for IHC, 68102-1-Ig, Proteintech, USA), anti-p38 (1:500 for western blotting, 1:1000 for IHC, WLH3870, Wanleibio, China), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1000 for western blotting, ab8245, Abcam, UK), anti-beta-actin (β-actin) (1:1000 for western blotting, ab8226, Abcam, UK), anti-caspase-1 (1:500 for western blotting, GB11383, Servicebio, China), anti-ASC (1:1000 for western blotting, ab309497, Abcam, UK), anti-IL-1β (1:1000 for western blotting, ab254360, Abcam, UK), anti-mouse IL-18 (1:2000 for western blotting, GB114098, Servicebio, China), and anti-phospho-p38 (p-p38) (1:1000 for western blotting, 28796-1-AP, Proteintech, USA).

The CAFs and macrophages seeded on sterile coverslips in 24-well plates were washed thrice with PBS and fixed with 4% paraformaldehyde for 15 min. Then cells were permeabilized with 0.1% Triton X-100 for 15 min and incubated with 2% BSA for 1 h. Next, cells were incubated with primary antibodies anti-ANGPTL2 (1:200, cat. # 12316-1-AP, Proteintech), anti-α-SMA (1:100, cat. # ab240654, Abcam), anti-CD68 (1:200, cat. # 28058-1-AP, Proteintech), anti-SPP1 (1:200, cat. # sc-21742, Santa Cruz), anti-vimentin (1:200, cat. #ab92547, Abcam), anti-KRT20 (1:100, cat. # ab76126, Abcam), or anti-Desmin (1:200, cat. # ab32362, Abcam) at 4°C overnight. After washing thrice, the corresponding secondary antibody goat anti-rabbit IgG H&L (Alexa Fluor^® 647) (1:400, cat. #ab150079, Abcam) or goat anti-mouse IgG H&L (Alexa Fluor^® 488) (1:400, cat. #ab150113, Abcam) was added, and the cells were incubated at room temperature for 1 h. Nuclei were counterstained with DAPI (cat. #P0131, Beyotime). Images were obtained using a microscope (Imager. A2, ZEISS).