V900502 100ml

The tissue sections were first deparaffinized and rehydrated prior to boil in a 100°C citrate buffer water bath for 30 minutes and then washed with PBS and permeated with 0.5% triton^TN X‐100 (V900502‐100ML, Sigma) in PBS followed by 3% fetal bovine serum (A602440, BBI, China) for 1 hour before incubating with primary antibodies overnight at 4°C. On the next day, sections were washed with PBS and incubated with the secondary antibodies for 1 hour at room temperature. The nuclei were stained with Hoechst 33324. The antibodies against proliferating cell nuclear antigen (PCNA) (ab29, Abcam) and Cytokeratin 14 (ab181595, Abcam) were used as primary antibodies. Secondary antibodies were Alexa 488‐conjugated‐goat anti‐rabbit IgG (ab150077, Abcam) and Alexa 647‐conjugated‐goat anti‐mouse IgG (ab150115, Abcam). Images were taken by a laser‐scanning confocal microscope (Leica TCS SP8, Leica, Germany). We analyzed the PCNA⁺ keratinocytes near the edge of the wounds and calculated the proliferation ratio.

Isolated lung tissues were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 4‐μm‐thick sections, and stained with haematoxylin and eosin. Infiltrating neutrophils were revealed by immunofluorescence using anti‐mouse Ly‐6G/Ly‐6C (Gr‐1) antibody (BioLegend) followed by Alexa 488‐conjugated‐goat anti‐rabbit IgG (Abcam) secondary antibody.
hMSCs were fixed with 4% paraformaldehyde for 15 min, then washed with PBS and permeated with 0.5% Triton X‐100 (V900502‐100ML, Sigma) in PBS for 15 min. The nuclei were stained with Hoechst 33324 (H3570, Thermo Fisher Scientific). The antibodies against Vimentin (ab92547, abcam), HP1α (ab109028, abcam), Lamin B1 (ab16048, abcam), Lamin A/C (ab8984, abcam), H3K9Me2/3(5327, CST), SP100 (ab167605, abcam), and PML (ab179466 and ab96051, abcam) were used as primary antibodies. Secondary antibodies were Alexa 488‐conjugated‐goat anti‐rabbit IgG (Abcam) and Alexa 555‐conjugated‐goat antimouse IgG (Thermo Fisher Scientific). Images were taken by a laser‐scanning confocal microscope (Leica TCS SP8, Leica, Germany).

The eyeballs were fixed in 4% paraformaldehyde for 24 h at room temperature, dehydrated with 30% sucrose before transferred into OCT medium (4583, SAKURA) and then frozen at −20 °C. The tissues were sectioned into 8 μm in thickness and placed on glass slides. The tissues mounted on slides were then washed with ddH₂O for 10 min to remove OCT. For hematoxylin-eosin staining, slides were stained with hematoxylin (MINDEL GTS-1096) for 5 min, then stained with eosin (G1100, Solarbio) for 90 s. For immunofluorescence staining, slides were treated with 0.5% Triton (V900502-100ML, Sigma) for 15 min followed by 10% donkey serum for 1 h before incubating with primary antibodies overnight at 4 °C. On the next day, sections were washed with 0.1% Tween 20 in PBS (PBST) and incubated with the secondary antibodies for 1 h at room temperature. The nuclei were stained with Hoechst 33324 (H3570, Thermo Fisher Scientific). The antibodies (Abcam) against CD31, α-smooth muscle actin, Ly6G and MPO were used as primary antibodies. Secondary antibodies were Alexa 488-conjugated-goat anti-rabbit IgG (Abcam), Alexa 594-conjugated-goat anti-rat IgG (Abcam), and Alexa 555-conjugated-goat anti-mouse IgG (Thermo Fisher Scientific). Images were captured under a laser-scanning confocal microscope (Leica TCS SP8, Leica).