Diaminobenzidine solution

The animals underwent anesthesia before receiving an intracardial infusion of 0.9% saline, followed by 4% PFA in PBS. The brains were carefully removed thereafter. For c-Fos labeling, the same methods used for GABA (Sigma) labeling were utilized, with the exception that the main antibody was swapped with an antibody specific to c-Fos (Cell Signaling Technology, Boston, USA), and the secondary antibody was replaced with goat anti-rabbit Alexa Fluor 488 (Jackson ImmunoResearch, West Grove, USA). Finally, all slices were washed in 0.1 mol/L PBS and cover-slip mounted in an antifade aqueous mounting medium containing DAPI (Roche, Basel, Switzerland).
For anti-CD31 labeling, sections were incubated overnight at 4°C and then treated with the secondary antibody, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:1000, Wuhan Boster Biological Technology, Wuhan, China), for 30 min at room temperature. Each segment was then immersed in a diaminobenzidine solution (ZSGB-Bio, China) and the nuclei were counterstained with hematoxylin. Subsequently, the sections were photographed using a microscope.

Immunohistochemistry (IHC) assay was carried out according to the reported method (Wang et al., 2015 (link)). Briefly, small intestine samples were de-waxed with xylene, rehydrated by graded alcohols, and then air-dried. Antigen retrieval was accomplished with 0.01 mol/l citrate buffer (pH 6.0) boiling for 15 min, followed by 3 PBS rinses. The slides were next incubated at 4°C for 12 h with PEDV mAb 3F12 (diluted 1:500). After three washes with PBS, sections were flooded and incubated for 1 h at 37°C with 1:100 diluted HRP-goat anti-mouse IgG (Biomedical Technologies, United States). After being washed three times in PBS, the slides were incubated for 5 min at room temperature in Diaminobenzidine solution (ZSGB-BIO, Beijing, China).

Immunohistochemical measurements of angiogenesis were performed on days 7 and 10 postsurgery. Mouse wounds were used to make paraffin sections as above. Tissue sections were deparaffinized, rehydrated, and pretreated for heat-mediated antigen retrieval. Sections were then incubated with anti-mouse CD31 antibody (1 : 1000, ab124432, Abcam, Britain) followed by incubated with biotinylated secondary antibodies (ZSGB-Bio, China) at room temperature for 30 min. All sections were colored by diaminobenzidine solution (ZSGB-Bio, China), and the nuclei were counterstained with hematoxylin (Beyotime, China). Then, the sections were photographed using a microscope.