Ab117644

After MRI, lesions were excised, embedded in paraffin, and cut into 5 μm sections. Sections were deparaffinized in xylene, rehydrated, and antigen retrieval was performed with 10mM sodium citrate buffer (pH 6) for 20 min. Sections were quenched, blocked, and incubated overnight with primary antibodies against myelin basic protein (MBP, Dako A0623, 1:500), CD68 (microglia/macrophages; CellSignaling #76437, 1:500), iNOS (inducible nitric oxide synthase; Novus NB120‐15203, 1:300), Ferritin (abcam ab75972, 1:100), CD206 (abcam ab117644, 1:200), and MerTK (tyrosine‐protein kinase MER; abcam ab52968, 1:500). Tissue was processed with the appropriate biotinylated secondary antibodies and avidin/biotin staining kit with diaminobenzidine (DAB) as chromogen (Vector Laboratories ABC Elite Kit and DAB Kit). Negative controls included isotype‐controls and absence of immunolabeling in tissues that do not express the above antigens. DAB‐enhanced Perls’ Prussian blue was used to detect ferric iron. Slides were immersed in 4% ferrocyanide/4% hydrochloric acid for 30 min in the dark, and staining was enhanced through incubation with DAB for 30 min at room temperature. After staining, all sections were rinsed, dehydrated, cover‐slipped, and digitized using a Mirax digital slide scanner.

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.