Goat anti rabbit igg cy3

Mice were transcardially well-perfused with 0.9% saline infusion followed by 4% paraformaldehyde in 0.1 M phosphate buffer. The tissues of the colons and the brains were dissected and immediately placed in 4% paraformaldehyde for 24 h, and next in 4% paraformaldehyde containing 30% sucrose. The brain and the colon tissues were embedded in paraffin and sliced into 3-μm-thick and 5-μm-thick slides, respectively. Briefly, the slides went through antigen retrieval using sodium citrate solution (pH 6.0). After being blocked with 3% bovine serum albumin (Servicebio, G5001), the slides were incubated with primary antibodies at 4 °C overnight. The primary antibodies include the following: anti-F4/80 (Servicebio, GB113373, 1:5000 dilution), anti-ZO-1 (Servicebio, GB111981, 1:200 dilution), anti-IL-23 (Wanleibio, WL01655, 1:200 dilution), and anti-Iba-1 (Abcam, Ab178847, 1:500 dilution). Then, the appropriate secondary antibody goat anti-rabbit IgG-Cy3 (Servicebio, GB21303, 1:300 dilution) was used to detect the corresponding primary antibodies. DAPI solution (Servicebio, G1012) was used to detect nuclei. The representative images were captured by a fluorescence microscope (Nikon Eclipse C1, Tokyo, Japan). The quantification of positively stained cells was conducted using ImageJ.

Cells were cultured on coverslips, washed in cold PBS, fixed in 95% ethanol for 30 min, and permeabilized in 0.3% Triton X-100 (Solarbio, Beijing, China) for 15 min. Fixed cells were blocked in PBS containing 5% goat serum (ZSGB, Beijing, China) at 37 °C for 1 h. Cells were incubated with the primary antibody CD133 (1:1000, #66666-1-lg, Proteintech, Wuhan, China) and Jagged1 (1:400, #bs-1448R, Bioss, Beijing, China) at 4 °C overnight. The secondary antibody goat anti-rabbit IgG (Cy3, #GB303, Servicebio, Wuhan, China) was added on cells which were washed three times at 37 °C in the dark for 1 h. DAPI (Solarbio, Beijing, China) was added on cells for 5 min. The fluorescent signal was captured using a TS100 inverted fluorescence microscope (Nikon, Japan).