Dapi dihydrochloride

MLO-Y4 cells were grown on glass coverslips in 24-well plates and treated when they reached 60% confluence. The cells were divided into four groups: a) control group treated with culture media only, b) high glucose group treated with culture media containing 20 mM glucose; c) inhibitor group treated with mitogen-activated protein kinases (MAPK) PD98059 (20 μM) or extracellular regulated protein kinases, (ERK) inhibitor U0126 (25μM) (MedChemExpress, Princeton, NJ, USA), and d) inhibitor (PD98059 or U0126) plus high glucose (20 mM) group. Cells were treated for 24 h, washed with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature, and permeabilized using 0.1% Triton X-100 in PBS for 10 min at room temperature. The cells were blocked with 10% goat serum for 2 h at room temperature and incubated with a primary antibody against Cx43 at a 1:50 dilution overnight at 4°C. The coverslips were washed with PBS, then incubated with Cy3-labeled goat anti-rabbit secondary antibody (SA000092, Proteintech, Rosemont, IL, USA) in the dark at room temperature for 1 h. Nuclei were co-labeled using DAPI dihydrochloride (Beyotime) for 5 min at room temperature. The coverslips were mounted on slides with anti-fade mounting medium (Beyotime), and images were collected using a fluorescence microscope (Eclipse Ci, Nikon, Japan).

At different times (3, 7, and 14 days), the wound tissue was taken and fixed with 4% paraformaldehyde, paraffin-embedded, sliced into approximately 5 ​μm sections, and finally stained with H&E, Masson's trichrome and Picrosirius red to evaluate wound healing. For immunofluorescence evaluation, the deparaffinized and rehydrated sections of the treated wound tissue (7, 14 days) were combined with anti-CD31 antibody (ab199012, 1:200) and anti-α smooth muscle actin antibody (ab7817, 1:200) and incubated overnight at 4 ​°C. Then, the corresponding fluorescently labeled secondary antibodies (Alexa Fluor/ab150077 and ab150115, 1:400) were used. Similarly, the wound tissue (14 days) was frozen and sectioned and incubated with ethidium dihydrogen (DHE, 2 ​μM) at 37 ​°C for 30 ​min to measure the overall superoxide content of the sample. The nucleus was stained with hematoxylin or DAPI dihydrochloride (C1006, Beyotime). Finally, the tissue sections and immunofluorescence sections were observed with an ordinary microscope (NL CD500, China) and an inverted fluorescence microscope (IFM Nikon Corporation, Japan), respectively.