Ihc wash buffer

Briefly, fresh tissues were fixed via immersion in 4% paraformaldehyde (Sigma-Aldrich) at RT for 30 min. The tissues were then subjected to gradient ethanol dehydration, embedded in paraffin, sectioned (thickness, 6 μm), and dewaxed in xylene. Next, the tissue sections were blocked at 37°C for 30 min in immunohistochemistry (IHC) blocking solution (Beyotime Biotechnology Co., Ltd., Zhejiang, China). After removal of the blocking solution, the tissue sections were washed 3 times at RT for 5 min each using IHC wash buffer (Beyotime Biotechnology Co., Ltd.). Subsequently, the tissue sections were incubated with primary antibodies (Table II) at 37°C for 45 min. After removal of the primary antibodies, the tissue sections were washed 3 times at RT for 5 min each using IHC wash buffer (Beyotime Biotechnology Co., Ltd.). The tissue sections were then incubated with secondary antibodies (Table I) at 37°C for 45 min. The secondary antibodies were subsequently discarded, and the tissue sections were washed 3 additional times at RT for 5 min each using IHC wash buffer (Beyotime Biotechnology Co., Ltd.). Finally, the tissue sections were mounted using neutral resin (Sigma-Aldrich) or fluorescence mounting medium (Sigma-Aldrich).