Anti n2icd

In brief, fresh colon tissues were fixed in 4% paraformaldehyde at 4 °C overnight, then subjected to paraffin imbedding sections. For H&E Staining, the sections were stained with haematoxylin and dehydration in graded alcohols and xylene. For immunolabeling, the sections were incubated with indicated primary antibodies: anti-CD3 [1:400, 85061; Cell Signaling]; anti-Ki67 [1:200, GB13030-2; Servicebio], anti-F4/80 [1:200, GB11027; Servicebio], anti-ChgA [1:400, ab45179; Cell Signaling] anti-CAI [1:200, SC39349; Santa Cruz], anti-Lysozyme [1:300, ab108502; Abcam], anti-N2ICD [1:200, YC0069; Immunoway], overnight at 4 °C in the dark. Then, the sections were incubated with either HRP–conjugated Goat anti-Rabbit IgG (1:200, G1215; Servicebio) or HRP–conjugated Goat anti-Mouse IgG (1:200, G1214; Servicebio) for 50 min at 25 °C. The subsequent detection was performed using the standard substrate detection of DAB. TUNEL assay kit was purchased from Abcam (ab66110). Images were taken by using Leica DM6 B Upright Microscope.

Paraffin sections of colon tissues were firstly dehydrated in gradient ethanol and blocked by 5% BSA in 0.2% TritonX-100/PBS, then incubated with specific primary antibody overnight at 4 °C: anti-GP73 [1:100, sc-48011; Santa Cruz],anti-N2ICD [1:100, YC0069; Immunoway], followed by incubation of secondary antibodies accordingly: Cy3 conjugated Goat Anti-Rabbit IgG (H+L) [1:300, GB21303; Servicebio] or FITC conjugated Donkey Anti-Goat IgG (H+L)[1:200, GB22404; Servicebio], and preserved in mounting medium with DAPI. The fluorescence was analyzed using Olympus Confocal Microscope.