Za 0550

Briefly, 3 μm sections were obtained from formalin-fixed, paraffin-embedded (PPFE) cell block preparations. Heat-induced epitope retrieval was applied with 0.02 M concentration of citrate buffer (pH 6.0) in a heater for 10 min. Immunostaining was performed with mAbs against MGA at 4 °C overnight in a dark humid chamber. After washing the slides in PBS, a streptavidin-biotin system was applied. DAB (3, 3′-diaminobenzidine) was used for color development. The sections were counterstained with hematoxylin, dehydrated and mounted with neutral gum. Appropriate positive and negative control slides were prepared.
For MGA and CD34 double staining, the sections were incubated with a 1:1 volume mixture of mouse mAb(MJF656) and Rabbit CD34 mAb(ZA0550, ZSGB-BIO, Beijing) in a buffer (1% bovine serum albumin in PBS) at 4 °C overnight in a dark humid chamber. Sections were firstly incubated with HRP conjugated goat anti-rabbit antibody (ZB2301, ZSGB-BIO, Beijing) for 0.5 h at room temperature and DAB were used for color development. Then, the sections were incubated with AP conjugated goat anti-mouse secondary antibody (SAP-9102, ZSGB-BIO, Beijing) followed by color development with AP-red (ZLI-9042, ZSGB-BIO, Beijing). Finally, sections were counterstained with hematoxylin, dehydrated and mounted with Clearmount (ZLI9553, ZSGB-BIO, Beijing).

For evaluation of the percent of CD34⁺ PD-L1⁺ vessels in tumor tissues. All patient sections were double-stained with anti-CD34 antibody (ZA-0550, ZSGB-BIO) and anti-PD-L1 antibody (66248-1-lg, Proteintech) using a double-staining kit (DS-0001, ZSGB-BIO). First, the most-abundant sites of blood vessels were selected under fourfold magnification of microscope, and four sites were randomly selected under a 40-fold objective lens, and the proportion of PD-L1⁺ CD34⁺ cells in all CD34⁺ vessels was calculated and averaged. The average percentage was used as the final result for each section. To evaluate the positive expression of VEGFA (ab1316, Abcam) and hypoxia-inducible factor α (HIF-1α) (ab113642, Abcam), stained sections were digitally analyzed at ×400 resolution using an Olympus BX-UCB. HIF-1α and VEGFA expression were scored by the H-score system, respectively. H score calculation method is based on previous literature^{15 (link)}.