Donkey serum

Co-cultures of enteric neurons and muscularis macrophages were fixed with 4% Paraformaldehyde (PFA) solution (#15710; Electron Microscopy Science). The samples were blocked and permeabilized in 10% donkey serum (#D9663; Sigma), 0.1% Triton in PBS. Primary antibodies were incubated in PBS containing 2% donkey serum and 0.1% Triton (Sigma Aldrich, Cat. #X-100) at 4°C overnight, followed by secondary antibody for 1 hour at room temperature and stained with DAPI (#D1306; Invitrogen) for 5 minutes. Primary antibodies used in this study included CD11b (#101212, 1:200; BioLegend) and IL1β (ab254360, 1:200; Abcam). Secondary antibody donkey anti-rabbit 488 (A21206, 1:200; Invitrogen) on a Keyence BZX-700 All-In-One Microscopy system, and the fluorescence of IL1β in muscularis macrophages, was quantified by ImageJ in a 10 mm2 area from each sample.

Cell coverslips were prepared 48 h after vector or recombinant protein treatment and were fixed using paraformaldehyde for 15 min and washed using PBS. Next, 200 µl of the corresponding primary antibody (1:500; cat. no. sc-390242; Santa Cruz Biotechnology, Inc.) diluted with 1% donkey serum (Sigma-Aldrich; Merck KGaA) was dripped onto each coverslip, which was then incubated at 4°C overnight. After incubation, the coverslips were washed with PBS and incubated at 4°C for 1.5 to 2 h, after pipetting 200 µl secondary antibody (1:500; cat. no. sab4600003; Sigma-Aldrich; Merck KGaA) diluted with 1% donkey serum. Then, the coverslips were washed with PBS, stained with DAPI combined with FITC (Sigma-Aldrich; Merck KGaA) and incubated at 4°C for 5 min. The treated cells were moved from the multi-well plate onto microscopic slides and spun. Next, the coverslip was placed onto an object slide, 5 µl fluorescence protection solution was pipetted onto each slide, and the whole system was sealed with cover glass. Finally, the sample was examined under confocal laser scanning microscopy (magnification, ×100; FV1000; Olympus Corporation) and imaged.

Immunofluorescence (IF) staining was used to detect keratinocyte proliferation and differentiation. For IF staining of primary keratinocytes, cells were briefly fixed with 4% paraformaldehyde (4% PFA) for 15 min, rinsed with phosphate-buffered solution (1×) (PBS, 1^st Base, Singapore), blocked with 10% donkey serum (Sigma, USA) and incubated at 4°C overnight with primary antibodies. Slides were then rinsed and incubated with secondary antibodies and Hoechst (Sigma, USA) for 60 min, rinsed with PBS and mounted with Prolong-Gold Anti-fade reagent (Invitrogen, USA). Confocal images were captured on the Zeiss LSM700 (Zeiss, Germany).
For IF staining of paraffin sections, standard dewaxing and rehydration steps were performed. Antigen retrieval was performed using citrate buffer pH6 (Novus Biologicals, USA) and 2100 Antigen Retriever (ProteoGenix, France), followed by Proteinase K (Sigma, USA) digestion for 15 min at room temperature. Sections were rinsed with PBS and blocked with donkey serum (Sigma, USA), and then incubated with primary antibodies overnight at 4°C. Next, the slides were rinsed in PBS-T (0.05% Tween-20, BioBasic, Singapore) and incubated with secondary antibodies and Hoechst (Sigma, USA) for 60 min, and then rinsed with PBS and mounted with Prolong-Gold Anti-fade reagent (Invitrogen, USA). Confocal images were captured on the Zeiss LSM700 (Zeiss, Germany).