Detection of VISTA/B7-H5/PD-1H by Western Blot. Western blot shows lysates of mouse platelets. PVDF membrane was probed with 1 µg/mL of Sheep Anti-Mouse VISTA/B7-H5/PD-1H Antigen Affinity-purified Polyclonal Antibody (Catalog # AF7005) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016 ). A specific band was detected for VISTA/B7-H5/PD-1H at approximately 30 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 8 .
Gi24/VISTA/B7-H5 in D3 Mouse Cell Line. Gi24/VISTA/B7-H5 was detected in immersion fixed D3 mouse embryonic stem cell line using Sheep Anti-Mouse Gi24/VISTA/B7-H5 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF7005) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Sheep IgG Secondary Antibody (red; Catalog # NL010 ) and counterstained with DAPI (blue). Specific staining was localized to cell surfaces and cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips .
Detection of VISTA/B7-H5/PD-1H in HEK293 Human Cell Line Transfected with Mouse VISTA and eGFP by Flow Cytometry. HEK293 human embryonic kidney cell line transfected with either (A) mouse VISTA or (B) irrelevant transfectants and eGFP was stained with Sheep Anti-Mouse VISTA/B7-H5/PD-1H Antigen Affinity-purified Polyclonal Antibody (Catalog # AF7005) followed by Allophycocyanin-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # F0127 ). Quadrant markers were set based on control antibody staining (Catalog # 5-001-A ). View our protocol for Staining Membrane-associated Proteins .