Cy5 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.

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).