Shandon coverplates

Frozen tissue was embedded in Frozen Section Medium (Azer Scientific), cut on a cryostat (5 um), and mounted onto Superfrost Plus glass microscope slides. Tissue was fixed on slides with acetone (−20 °C) and endogenous peroxidase activity was blocked with 0.6 % hydrogen peroxide. Slides were placed on Shandon coverplates™ (Thermo Fisher) using 0.2 % Triton-× 100/PBS and placed in a Sequenza™ slide rack (Thermo Fisher). Slides were incubated with Biotin conjugated rat anti-mouse CD8 (eBioscience, 1:50 in 1%BSA/PBS), or diluent as negative control, and then with Vectastain Elite ABC (Vector Laboratories, Burlingame, CA), developed with Vector DAB Peroxidase substrate (Vector Laboratories) and mounted with Permount (Thermo Fisher).

Tissue sections were deparaffinized in xylene and rehydrated through graded ethanol. For antigen retrieval slides were steamed for 30 minutes in EnVision FLEX Target Retrieval Solution (pH 6.1) (Dako). Antibody incubations were done overnight at 4 °C in Shandon coverplates (Thermo Fisher Scientific). For detection, HiDef HRP polymer system (Sigma-Aldrich) was used followed by incubation with DAB + chromogen (DAKO) according to manufacturer’s instructions. Slides were counterstained with Mayers hematoxylin for 1 minute. All immunohistochemical procedures were undertaken in one batch. The antibody for p65 was from Cell Signaling Technology.