G1235

Tissue microarray of osteosarcoma specimens (L714901) was obtained from Biotech Company (Xian, China) and used to explore further the association of TYROBP expression with CD8⁺ T cell in the osteosarcoma tumor microenvironment.
Multiplex immunohistochemistry was conducted using the sequential staining cycles as follows. In brief, formalin-fixed, paraffin-embedded osteosarcoma tissue sections were deparaffinized and then underwent microwave treatment in citrate for antigen retrieval. Then, they were blocked with 10% normal goat serum and incubated overnight with primary antibodies: mouse anti-CD8 antibody (1:100, ab17147, Abcam) and rabbit anti-TYROBP antibody (1:200, ab124834, Abcam). Next, sections were incubated with the corresponding horseradish peroxidase-conjugated second antibodies (Abcam, CN) for 30 min at room temperature. The antigenic binding sites were visualized using the tyramide signal amplification dye. Fluorescein isothiocyanate-tyramide (1:1,000, G1235, Servicebio) and Cy3-tyramide (1:1,000, G1235, Servicebio) were applied to each antibody.

A fluorescent double staining kit (G1235; Servicebio, Wuhan, China) based on tyramide signal amplification was performed in accordance with the manufacturer’s instructions. Primary antibodies for histological staining included CD31 (dilution 1 : 5000; GB13428; Servicebio) and PDGFR‐β (dilution 1 : 500; #3169; Cell Signaling, Danvers, MA, USA).
Purified pericytes were fixed, permeabilized, and subsequently blocked with BSA for 1 h. Cells were then incubated with antibodies against α‐SMA (dilution 1 : 100; ab7817; Abcam, Cambridge, UK), PDGFR‐β (dilution 1 : 100; #3169; Cell Signaling), neuron‐glial antigen 2 (NG2) (dilution 1 : 100; sc‐53389; Santa Cruz Biotechnology, Dallas, TX, USA), and CD13 (dilution 1 : 100; sc‐13536; Santa Cruz Biotechnology) overnight at 4 °C. The next day, the cells were washed and incubated with secondary Alexa Fluor 488 anti‐rabbit antibody (dilution 1 : 1000; 4412; Cell Signaling) or Alexa Fluor 594 anti‐mouse antibody (dilution 1 : 1000; 8890; Cell Signaling) for 1 h when protected from light. Nuclear staining was performed with 4′,6‐diamidino‐2‐phenylindole. The images were captured by high‐content analysis (Operetta; PerkinElmer, Waltham, MA, USA). Image analysis and fluorescence quantification were performed using columbus (PerkinElmer) and imagej (NIH, Bethesda, USA).