Ab187048

Normal and wounded mouse skin tissues were harvested and fixed overnight with 4% paraformaldehyde. Then, the tissue was washed, dehydrated, and embedded in paraffin. Next, 4-μm tissue sections were prepared for Masson trichrome staining (Sigma-Aldrich) and immunohistochemical staining (IHC) according to well-established protocols. Masson trichrome staining was used to analyze total collagen accumulation according to the manufacturer’s instructions. For IHC staining, after deparaffinization, rehydration, and antigen retrieval (sodium citrate buffer, pH 6.0), the samples were incubated with CD26 primary antibody (1: 1000, ab187048, Abcam) and corresponding secondary antibodies (Maxim, China) as described previously [15 (link)]. The negative controls group underwent incubation without primary CD26 antibody.

Kidney slides were incubated overnight at 4°C with primary antibodies anti-DppD (1:100, ab187048, Abcam, UK), anti-Claudin-2 (1:100, Product#51-6100, Invitrogen, USA), and anti-α-SMA (1:200, BM0002, BOSTER, China). Dipeptidyl peptidase (DppD) was used as a marker of PTs. Negative controls were incubated with phosphate-buffered saline (PBS). At room temperature, NRK49F cells cultured on coverslips were fixed with 4% paraformaldehyde for 5 minutes, permeabilized with 0.1% TritonTMX-100 for 10 minutes, and blocked with 2% BSA for 45 minutes. The cells were labeled with antibodies against PCNA (1:200, 10205-1-AP; Proteintech, Wuhan, China), α-SMA (1:200, BM0002, BOSTER, China), and anti-Claudin-2 (1:100, Product#51-6100, Invitrogen, USA). Secondary antibodies Alexa Fluor^® 488 donkey anti-rabbit (Product#A32790, Invitrogen, USA), Alexa Fluor^® 488 donkey anti-mouse (Product#A-11029, Invitrogen, USA) were used at a 1:300 dilution in vivo and 1:800 dilution in vitro, respectively. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI)(Sigma-Aldrich) according to the manufacturer’s instructions. Images were captured by fluorescence microscopy (Ni-V, Nikon, Japan), and photographs were recorded.