Tcs sps microscope

For tissue samples, paraffin-embedded intestinal sections were dewaxed in xylene and rehydrated through gradient alcohols. The masked antigens were retrieved by 0.01 M citrate buffer solution. After being blocked with 5% bovine serum albumin (BSA, Sigma-Aldrich), the tissue sections were stained with anti-Muc2 (Santa Cruz Biotechnology), anti-Lysozyme (Abcam) and anti-Olfm4 (Cell Signaling Technology). Immunohistochemistry was analyzed by goat anti-rabbit IgG conjugated to horseradish peroxidase (Servicebio). For immunofluorescence, signals were detected by goat anti-rabbit Alexa Fluor 488 conjugate (Abcam), and then counterstained with DAPI (Abcam). For intestinal organoids, after being fixed by 4% paraformaldehyde (Servicebio) and permeated by 0.5% Triton X-100 (Beyotime), the organoids were blocked by 2% BSA, incubated with anti-GFP (Abcam) at 4 °C overnight, and stained by goat anti-rabbit IgG Alexa Fluro 488 conjugate (Cell Signaling Technology) for 1 h, then 1 mg/mL DAPI (Sigma-Aldrich) was used to stain the nucleus. The images were captured and analyzed by Leica TCS SPS microscope and Leica Application Suite X software.

Cell death was detected with an in-situ cell death detection kit (Roche) following the manufacture’s instruction. Briefly, paraffin-embedded intestinal sections were dewaxed in xylene and rehydrated through gradient alcohols. The slides were then incubated with 0.1% Triton X-100 in 0.1% sodium citrate buffer for 8 minutes followed by PBS washing. Next, the samples were incubated with the TUNEL reaction mixture in a humidified chamber at 37 °C for 60 min protected from light. This was followed by PBS washes and incubation with 1 mg/mL DAPI (Sigma-Aldrich) for 10 min. Fluorescence microscopy was performed using a Leica TCS SPS microscope.