Ab111250

The excised VX2 tumor tissues were fixed in 4% (w/v) paraformaldehyde for 24 h at room temperature, embedded in paraffin and sliced into 4 µm sections. Tissue sections were deparaffinized in xylene and hydrated gradually through graded alcohol and processed with Tris/EDTA buffer solution (pH 9.0) and high pressure cooker, >120°C for 3 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 15 min. The slides were then blocked with 10% goat serum (AR0009, Boster Biological Technology Co., Ltd., Wuhan, China) for 2 h at room temperature, and primary antibodies of CD206 (1:25, ab117644; Abcam, Cambridge, UK) and CD163 (1:50, ab111250; Abcam) were applied overnight at 4°C. The biotin-labeled secondary antibody (KIT-7710; Maixin Biotech Co., Ltd., Fuzhou, China) was applied for 1 h at room temperature. The sections were stained at room temperature with diaminobenzidine tetrahydrochloride (Maixin Biotech Co., Ltd.) for 50 sec and finally counterstained with hematoxylin for 8 min. Sections stained in the absence of primary antibodies were used as negative controls.
Five slides from each animal were examined under light microscopy. The proportion of positive cells was estimated among the total number of the cells by observing five random fields at ×400 magnification.