Fitc tsa

We obtained the tissue microarray from the Outdo Biotech company (HOrg-C110PT-01, Shanghai, China) and the ethics was approved. Briefly, the paraffin sections of pan-cancer samples were deparaffinized and then were blocked with 3% H2O2 and 2% BSA after antigen retrieval. Anti-PDIA5 antibody (Rabbit, 1:100, Proteintech, China), anti-CD68 antibody (Rabbit, 1:3000, Servicebio, China), and anti-CD163 antibody (Rabbit, 1:3000, Proteintech, China) were sequentially applied, followed by horseradish peroxidase-conjugated secondary antibody (GB23301 and GB23303; Servicebio, China) incubation and tyramide signal amplification (TSA) (CY5-TSA, CY3-TSA, and FITC-TSA; Servicebio, China) incubation. And then, 4’,6-Diamidino2-phenylindole dihydrochloride (DAPI) was applied for the nuclei staining. The stained slides were visualized for multispectral images under the Pannoramic Scanner (3D HISTECH, Hungary). DAPI glows blue by UV excitation wavelength 330-380nm and emission wavelength 420nm in the fluorescence spectra. CY5, FITC, and CY3 glow pink, green, and red. The excitation wavelength was 608-648nm, 465-495nm, and 510-560nm, respectively, with an emission wavelength of 672-712nm, 515-555nm, and 590nm.

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.

The Histone H3, ECL, FITC-TSA, CY3-TSA, DAPI, and blueback fluid were purchased from servicebio Co., Ltd. (Wuhan, China). The HRP-labeled goat anti-mouse secondary antibody and HRP-labeled goat anti-rabbit secondary antibody were purchased from Wuhan Boster Biological Engineering Co., Ltd., (Wuhan, China), Fetal Bovine Serum, DMEM and penicillin-Streptomycin were purchased from GIBCO Co., Ltd., (Grand Island, NE, USA). All other chemicals used were of analytical grade.

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.

The gliomas chip was baked at 60°C for 60 min for deparaffinization, then the antigen was retrieved by EDTA retrieval buffer and blocked in 3% BSA. Next, the samples were permeabilized with 0.1% Triton X-100. The gliomas tissue microarray was incubated with the primary antibodies of ITGA5 (10569-1-AP, Rabbit, 1:200, Proteintech, China), CD68 (GB113150, Rabbit, 1:3,000, Servicebio, China), and CD163 (16646-1-AP, Rabbit, 1:3,000, Proteintech, China) separately, then followed by the incubation with secondary antibodies (GB23301, GB23303, Servicebio, China) and tyramide signal amplification (TSA) [FITC-TSA, CY3-TSA, and CY5-TSA (Servicebio, China)]. The antigen repair was applied repeatedly between the intervals of each dye. Subsequently, the microarray was incubated with 4’,6-Diamidino2-phenylindole dihydrochloride (DAPI). Microscopy detection was performed by the Pannoramic Scanner (3D HISTECH, Hungary).