Ab237726

Opal 7-colour kit (NEL811001KT, PerkinElmer) was used for mIF. TMAs were dewaxed and rehydrated. In the first step, the antigen was retrieved at 125 ℃ for 3 min and then cooled to room temperature (RT). Washed with TBST three times for 5 min, incubated in H₂O₂ for 10 min. Repeated washed and blocked with blocking buffer. The primary antibody, PDL-1 (ab237726, Abcam, 1:500, dye 480) was incubated at RT for 30 min. Slides were washed and an HRP-conjugated secondary antibody was incubated at RT for 10 min. TSA dye (1:100) was applied for 10 min after washes. The procedures were repeated six times using the following antibodies, CD3 (ab16669, Abcam, 1:200, dye 690; used as T lymphocyte cell marker [30 (link)]), CD8 (ab93278, Abcam, 1:100, dye 570; used as cytotoxic T cell marker [31 (link)]), CD56 (ab75813, Abcam, 1:500, dye 620; used as NK cell marker [32 (link)]), CD68 (ab213363, 1:1000, Abcam, dye 780; used as pan-macrophage marker [33 (link)]), programmed death-1 (PD-1) (ab237728, Abcam, 1:300, dye 520), programmed death ligand-1 (PD-L1) (ab237726, 1:500, dye 480) [34 (link)]. Secondary antibodies anti-mouse (NEF822001EA, PerkinElmer) or anti-rabbit (NEF812001EA, PerkinElmer) were used at a 1:1000 dilution.
With further analysis by the HALO system, we quantified the number of six immune targets and the spatial position relationship between them [35 ].

Eight glioma samples were fixed by immersion in 10% formalin solution and then embedded in paraffin. 10-µm thick tissue sections were deparaffinized and rehydrated (xylene × 2 for 10 minutes each, 100%, 95%, and 75% ethanol for 5 minutes each and deionized water for 5 minutes). The sections were incubated with 3% hydrogen peroxide in methanol for 10 minutes to quench peroxidase activity. Antigen retrieval was performed by boiling sections in 10 mM sodium citrate buffer (pH 6.0) for 10 minutes. After being rinsed with PBS, sections were blocked with normal goat serum for 20 minutes. The samples were incubated with primary anti-PD-L1 (1:200, ab237726, Abcam, Cambridge, UK) or anti-TGFβ antibody (1:200, BA0290, BOSTER, Wuhan, China) overnight at 4°C. The sections were then incubated with secondary antibody () for 30 minutes at room temperature. The staining was developed using 3,3’-diaminobenzidine (DAB) as substrate and counterstained with hematoxylin. The sections were developed using 3,3’-diaminobenzidine (DAB) as substrate and counter-stained with Mayer’s Hematoxylin. All sections were independently reviewed by two pathologists according to the WHO criteria.

Immunohistochemistry (IHC) staining was performed on these TMAs of PaCa tissues according to the standardized procedure. EDTA was used for antigen retrieval, and the primary antibodies were incubated overnight at 4 °C. The primary antibodies used in the research were as follows: anti-PD-L1 (1:100 dilution, Cat. ab237726, clone: CAL10, Abcam, Cambridge, UK), anti-B7-H3 (1:8000 dilution, Cat. ab219648, clone: EPR20115, Abcam, Cambridge, UK), anti-B7-H4 (1:50 dilution, Cat. ab252438, clone: EPR23665–20, Abcam, Cambridge, UK) and anti-CD8 (Ready-to-use, Cat. PA067, clone: 457F6F8, Abcarta, Suzhou, China). Notably, staining data for PD-L1 and CD8 of the HPanA120Su02 TMA was provided by Outdo BioTech (Shanghai, China). A total of 9 samples that fall off the HPanA120Su02 TMA during the IHC staining for B7-H4. Thus, 57 samples in the HPanA120Su02 TMA were used for further analysis. Antibody staining was visualized using diaminobenzidine and hematoxylin counterstain, and stained TMAs were scanned using Aperio Digital Pathology Slide Scanners.