Bl 95 1a2

Ten cases were selected based on previous assessment for K8.1, to include seven positive cases and three negative cases. These cases had been previously stained by immunohistochemistry (IHC) for KSHV LANA and K8.1, as part of an AIDS Malignancy Consortium (AMC) and AIDS Clinical Trials Group (ACTG) clinical trial, AMC066/A5263 (NCT01435018;PMID 32145827), and HIF2α IHC was added for this study. Immunophenotyping was performed on formalin-fixed, paraffin-embedded tissue sections on a Leica Bond III system using the standard protocol. Sections were pre-treated using heat-mediated antigen retrieval with Sodium-Citrate buffer (pH6, epitope retrieval solution 1) for 30 mins. The sections were then incubated with appropriate antibodies for 15 mins at room temperature and detected using an HRP-conjugated compact polymer system. 3,3′-Diaminobenzidine (DAB) was used as the chromogen. Sections were then counterstained with hematoxylin and mounted with micromount. The following antibodies were used for IHC: anti-LANA mouse monoclonal HHV-8 ORF73 (Leica, Catalog Number PA0050), anti-K8.1 mouse monoclonal (Advanced Biotechnologies, Clone 2A3, Catalog Number 13-213-100) and anti-HIF2a rabbit monoclonal clone BL-95–1A2 (Bethyl Laboratories, Catalog Number A700–003).