Kit 9720

Animals were euthanized and skin samples were excised, the tissues were fixed with 4% paraformaldehyde for 24 hours, then dehydrated with 70%, 75%, 85%, 95%, 100% ethyl alcohol, covered with paraffin, before sectioning and histological analysis. Blocks were cut to expose wounded tissue near the center of each wound and then cut into 4 μm thickness and stained with hematoxylin and eosin.
Number of blood vessels containing red blood cells was counted over the entire wound area using three fields per section. The thickness of the new skin evaluated as follows: The middle part of the neonatal skin wound and the two ends near the edge of the wound were, respectively, selected to measure the thickness and then calculated the average.
The tissue sections were first deparaffinized and rehydrated prior to boil in a 100°C citrate buffer water bath for 30 minutes (R20902, YUANYE, China) and then quenched of endogenous peroxidize using blocker (KIT‐9720, MXB, China) for 10 minutes before incubating with CD31(28 364, abcam, UK) antibody overnight in 4°C, to allow visualization of the immunostaining, sections were incubated with the anti‐rabbit‐biotinylated secondary antibody for 45 minutes, and then incubated with Streptaridin‐Peroxidase for 20 minutes and DAB (DAB‐2031, MXB, China) and counterstained with hematoxylin. The proportion of CD31 positive signals is calculated by ImageJ software.

After the sections were deparaffinized and rehydrated, the experiments were performed following the IHC kit instructions (kit-9720, MXB, China) for MMP13 (18165-1-AP; 1:200; Proteintech), Nrf2 (bs-1074R; 1:500; Bioss), P53 (A0263; 1:100; Abclonal), SLC7A11 (DF12509; 1:100; Affinity Biosciences), Col2a1 (28459-1-AP; 1:1000; Proteintech), p-NF-κB p65 (bs-5662R; 1:200; Bioss), and GPX4 (67763-1-Ig; 1:2000; Proteintech). A series of primary antibodies were added to the sections of the four groups, and then all sections were incubated in a wet box at 4 °C overnight. The following day, the sections were incubated at room temperature for 1 h with a secondary antibody, and a DAB solution (dab-0031, MXB, China) was added for visualization. Finally, after neutral resin blocking, they were photographed under a microscope. Five fields were randomly selected for each sample, and the expression of each index was examined and averaged for statistical analysis.

For immunohistochemistry staining, paraffin‐embedded brain sections (4 μm) were deparaffinized and rehydrated. Antigen retrieval was performed by boiling for 90 s in sodium citrate buffer (10 mM, pH 6.0) using a pressure cooker. Sections were stained with a immunostaining kit (KIT‐9720, MXB Biotechnologies). Tissue sections were incubated with antibodies for SIRT1 (1:250, Sigma, 07131), SIRT2 (1:200, ZEN Bioscience, 200474) overnight at 4°C. Antibody binding was detected using HRP‐conjugated anti‐rabbit or mouse secondary antibody and visualized using DAB Peroxidase Substrate kit (DAB‐0031, MXB Biotechnologies). The images were acquired using a microscope (Nikon, DS‐Fi2).