Dab peroxidase substrate kit

We performed IHC staining off of the target proteins in formalin-fixed, paraffin-embedded colon tissues. The antigens were retrieved by boiling the samples for 10 min in 10 mM citrate (pH 6.0) or EDTA antigen repair solution (pH 9.0) (ZSGB-BIO). The slides were stained with rabbit monoclonal antibodies (Cell Signaling) and then incubated with a peroxidase-conjugated secondary antibody. Finally, the signals were visualized with diaminobenzidine (DAB) peroxidase substrate kit (Servicebio).

The wax blocks were placed in a paraffin slicer for continuous sectioning, with each section having 4 μm thickness. HE staining was performed using HE staining solution (G1003, Servicebio, Wuhan, China). For IHC, paraffin tissue sections were deparaffinized with xylene and rehydrated with an alcohol gradient and water. Sections were incubated with primary antibodies IL17RA (Catalogue number: DF3602, diluted 1:100, Affinity), at room temperature for 1 h and biotin-labelled secondary antibodies for 30 min, and then stained with DAB peroxidase substrate kit (G1212, Servicebio, Wuhan, China). Finally, washed with water and counterstained with haematoxylin. The results were observed and photographed by an microscope (Eclipse C1, Nikon, Japan).