Ab252438

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.

Briefly, 3 μm thick formalin-fixed paraffin-embedded specimen slides were prepared for immunohistochemical analyses. The sections were deparaffinized and subjected to antigen retrieval, followed by peroxidase blocking with hydrogen peroxide (3%). After blocking with goat serum, sections were incubated with primary antibodies against B7-H3, B7-H4, HHLA2, CD8, and Foxp3 (anti-CD276 antibody: Abcam, ab105922; anti-B7H4 antibody: Abcam, ab252438; anti-HHLA2 antibody: Abcam, ab214327; anti-CD8α antibody: Abcam, ab237710; anti-Foxp3 antibody: Abcam, ab215206) overnight at 4 °C. After washing with phosphate-buffered saline (PBS), sections were incubated with secondary antibody for 1 hour. Visualization with 3,3′-diaminobenzidine (DAB) for 3 minutes. Nuclei were stained with Harris hematoxylin. Sections were dehydrated and then sealed with neutral gel.

To measure the levels of PD-L1, B7-H4 and CD8 in the CeCa samples, the multiplexed QIF was directly performed on the tissue section using a previously described protocol with simultaneous detection of DAPI [16 (link)]. The primary antibodies were as follows: anti-B7H4 (1:100 dilution, Cat. ab252438, Abcam, Cambridge, UK), anti-PDL1 (1:500 dilution, Cat. ab237726, Abcam, Cambridge, UK) and anti-CD8 (1:200 dilution, Cat. ab101500, Abcam, Cambridge, UK). The expression levels of B7H4 and PDL1 were evaluated according to the previous method. For CD8 staining, infiltration level was assessed by estimating the percentage of cells with strong intensity of membrane staining in the stroma cells. For stratification, the B7H4 and PDL1 levels were classified as high/low using the top 50-percentile of the cohort scores as stratification cut-point.