647 tsa

Paraffin sections of glioma tissues collected from Xiangya Hospital, Central South University, were deparaffinized. After antigen retrieval, sections were blocked with 3% H₂O₂ and 2% BSA. Different primary antibodies, CD68 (Mouse, 1 : 3000, AiFang Biological, Changsha, China), CD163 (Rabbit, 1 : 3000, Proteintech, Wuhan, China), were sequentially applied, followed by horseradish peroxidase‐conjugated secondary antibody incubation (PV6001, PV6002, ZSGB‐BIO, Beijing, China) and tyramide signal amplification (TSA; Fitc‐TSA, CY3‐TSA and 647‐TSA [Servicebio, Wuhan, China]). After labeling with the human antigens, nuclei were stained with 4′,6‐diamidino2‐phenylindole dihydrochloride (DAPI), and an antifade mounting medium was applied. Stained slides were scanned using the Pannoramic Scanner (3D HISTECH, Budapest, Hungary) to obtain multispectral images. Regarding fluorescence spectra, DAPI glows blue at a UV excitation wavelength of 330–380 nm and emission wavelength of 420 nm, CD163 glows red at an excitation wavelength of 594 nm and emission wavelength of 615 nm, and CD68 glows pink at an excitation wavelength of 608–648 nm and emission wavelength of 672–712 nm. Multispectral images were analyzed, and positive cells were quantified at a single‐cell level by caseviewer (C.V 2.3, C.V 2.0) and pannoramic viewer (P.V 1.15.3) image analysis software.

We obtained the tissue microarray from the Outdo Biotech company (HOrg-C110PT-01, total number of cases: 69 cases, total points: 110 points, Shanghai, China). The tissue microarray was approved by the Ethics Committee. Each tumor/normal tissue has three to eight cores (diameter 1.5 mm). The primary Abs were VSIR (Rabbit, 1:200, Proteintech, China), CD68 (Rabbit, 1:3000, AiFang biological, China), CD163 (Rabbit, 1:3000, Proteintech, China), CD8 (Mouse, 1:3000, Proteintech, China). The secondary antibody was horseradish peroxidase-conjugated secondary antibody incubation (GB23301, GB23303, Servicebio, China), and the tyramide signal amplification was TSA (FITC-TSA, CY3-TSA, 594-TSA, and 647-TSA (Servicebio, China)). Multispectral images were analyzed, and positive cells were quantified at single-cell levels by Caseviewer (CV 2.3, CV 2.0) and Pannoramic viewer (PV 1.15.3) image analysis software. Negative control procedures included the omission of the primary antibody.

We obtained the tissue microarray from the Outdo Biotechcompany (HOrg-C110PT-01, Shanghai, China) and the ethics was approved. The primary antibodies were CLEC5A (Rabbit, Sigma-Aldrich, US), CD68 (Rabbit, AiFang biological, China), CD163 (Rabbit, Proteintech, China), CD8 (Mouse, Proteintech, China). The secondary antibody was horseradish peroxidase-conjugated secondary antibody incubation (GB23301, GB23303, Servicebio, China), and the tyramide signal amplification was TSA [FITC-TSA, CY3-TSA, 594-TSA, and 647-TSA (Servicebio, China)]. Multispectral images were analyzed, and positive cells were quantified at single-cell levels by Caseviewer (CV 2.3, CV 2.0) and Pannoramic viewer (PV 1.15.3) image analysis software.