Tacha s hematoxylin

Chromogenic staining was performed (Liquid DAB + Substrate Chromogen, Dako), followed by counterstain with Tacha's hematoxylin (Biocare Medical) for Ki67, PD-L1, STING, γH2AX at Second Department of Pathology, University of Athens Medical School (Attikon University Hospital, Athens, Greece).

Tissue sections, 3–4 μm in thickness, were deparaffinized in CitriSolve (Decon Labs., King of Prussia, PA) and rehydrated by processing them through graded alcohol solutions. Antigen unmasking was accomplished by digesting tissue in 10 μg/ml of protein kinase for 15 min at room temperature (Fisher Scientific, Waltham, MA). Endogenous enzymes and non-specific background were blocked with Background Punisher (Biocare Medical, Pacheco, CA), followed by BLOXALL (Vector Laboratories, Burlingame, CA). The primary antibody (NBP1–42140; Novus Biologicals, Centennial, CO) was incubated on the tissue sections at a dilution of 1:100 for 1 h at room temperature. Subsequently, sections were sequentially incubated with an alkaline phosphatase-based detection polymer kit (MACH 4; Biocare Medical), and Warp Red (Biocare Medical). Sections were counterstained with Tacha’s hematoxylin (Biocare Medical) and mounted using a permanent mounting medium (EcoMount; Biocare Medical).

Tissues were fixed in 10% neutral buffered formalin, processed and embedded in paraffin, and sectioned at 4 μm thickness using standard histologic procedures. Slides were dewaxed using xylene and rehydrated with a graded series of ethanol using a DAKO Coverstainer (DAKO, Agilent Technologies). Antigen retrieval was performed in a high pH buffer using PT Link (DAKO, Agilent Technologies) at 95°C for 20 minutes. IHC was carried out using a DAKO Autostainer Link 48 platform (DAKO, Agilent Technologies). Briefly, slides were blocked for endogenous peroxidases and subsequently stained using the following primary antibodies: rabbit anti-mouse CD8 (D4W27 at 1:200), Rabbit anti-mouse CD45 (Cell Signaling Technology D3F8Q at 1:200), and Rabbit anti-mouse CD31 antibodies (Abcam EPR17259 at 1:500) in Da Vinci diluent (Biocare Medical). MACH2 Rabbit AP polymer (Biocare Medical) was used to detect primary antibodies, followed by detection using Enzo Red chromogen (Enzo Life Sciences). Slides were then counterstained with Tacha's Hematoxylin (Biocare Medical), dehydrated, and coverslipped using the DAKO Coverstainer. Once dried, slides were scanned using a 3D Histech Pannoramic MIDI II Scanner (3DHISTECH), then image/spatial analyses were performed using HALO software (Indica Labs).