Immunohistochemical blocking solution

On the third and tenth day after the first supernatant treatment, the mice were euthanized via cervical dislocation, and samples were harvested for tissue H&E staining and IF. Briefly, the tissues were fixed with 4% paraformaldehyde for 48 h and then covered with paraffin before sectioning and histological analysis. Blocks were cut into 4 μm thick sections and stained with H&E (YESEN). For IF assay, Briefly, Samples from the wound bed on day 3 were first dewaxed and rehydrated and then repaired antigen via boiling in a 100 °C citrate buffer water bath for 25 min. The tissue sections were blocked with immunohistochemical blocking solution (Beyotime, China) for 90 min. The primary antibodies used in this experiment were incubated at 4 °C overnight, as follows: F4/80 (Rat IgG, monoclonal, Abcam Cat. No.: ab6640), CD86 (Rabbit IgG, Polyclonal, SAB Cat. No.: 32223-2) and CD163 (Mouse IgG, monoclonal, GeneTex Cat. No.: ED2). Then incubated with the following secondary antibodies for 90 min at room temperature: Alexa 594-conjugated goat anti-Rat IgG (ab150160, Abcam), Alexa 488-conjugated goat anti-rabbit IgG (ab150077, Abcam) and Alexa 594-conjugated goat anti-mouse IgG (ab150116, Abcam). The nuclei were stained with DAPI (YESEN, China). Images were acquired using a laser-scanning confocal microscope (Carl Zeiss LSM880, Germany).

All fresh tissues were briefly soaked at room temperature and fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, USA) for 30 min. Ethanol gradient dehydration, paraffin embedding, slicing (thickness 6 µm), and dewaxing was performed using xylene. The tissue sections were incubated with an immunohistochemical blocking solution (Beyotime Biotechnology Co., Ltd., Zhejiang, China) at 37 °C for 30 min. The blocking solution was discarded, and an immunohistochemical cleaning solution (Beyotime Biotechnology) was added to rinse the tissue sections at room temperature 3 times for 5 min. The first antibody (Table 1) was added and incubated at 37 °C for 45 min. The antibody was discarded, and an immunohistochemical cleaning solution (Beyotime Biotechnology) was added to rinse the tissue for 5 minutes 3 times at room temperature. The secondary antibody (Table 1) was added and incubated at 37 °C for 45 min. The antibody was discarded before adding an immunohistochemical cleaning solution (Beyotime Biotechnology) to rinse the tissue at room temperature 3 times for 5 min. Finally, an immunofluorescence sealing solution (Sigma-Aldrich) was added to seal the slides.

Briefly, fresh tissues were immersed in 4% paraformaldehyde (Sigma-Aldrich) for fixation at room temperature for 30 min. The tissues were then dehydrated in an ethanol gradient, embedded in paraffin, sectioned (thickness: 6 μm), and immersed in xylene for dewaxing. Tissue sections were blocked with immunohistochemical blocking solution (Beyotime Biotechnology Co., Ltd., Zhejiang, China) at 37 °C for 30 min. The blocking solution was then discarded, and the sections were washed three times at room temperature for 5 min each with immunohistochemical washing solution (Beyotime Biotechnology). Then, primary antibodies were added and incubated at 37 °C for 45 min. After incubation, the antibody solution was discarded, and the sections were washed three times at room temperature for 5 min each with immunohistochemical washing solution (Beyotime Biotechnology). Then, secondary antibodies were added and the tissues were incubated at 37 °C for 45 min. After incubation, the antibody solution was discarded, and the sections were washed three times at room temperature for 5 min each with immunohistochemical washing solution (Beyotime Biotechnology). Finally, immunofluorescence blocking solution (Sigma-Aldrich) was added, and the sections were mounted.