Rm 9107

After heat-induced antigen retrieval at pH 6, formalin-fixed and paraffin-embedded tissue sections were incubated for 1 h at room temperature with a TRIM33 antibody (Sigma HPA004345, dilution 1:50), pH2AX antibody (Cell Signalling #9781, dilution 1:100), and a CD3 antibody (Thermo Scientific RM-9107, dilution 1:100), respectively. An anti-rabbit secondary antibody conjugated to AP (Polyview Plus AP (anti-rabbit) reagent, ENZO Life Sciences GmbH, Lörrach, Germany) was applied. The signal was visualized using alkaline phosphatase (Permanent-AP Red Kit, Zytomed Systems, Berlin, Germany) as a chromogen. pH2AX and CD3 stained sections were quantified manually using the Aperio ImageScope—Pathology Slide Viewing Software 12.4.6.

Tissues were incubated for 24 h in 4% phosphate-buffered formaldehyde solution (Roti-Histofix; Carl Roth), dehydrated, paraffin-embedded, and cut (4-μm-thick sections). For immunohistochemical staining, heat-mediated antigen retrieval was performed in citrate buffer at pH 6.0 (Dako) and sections were stained with monoclonal rabbit anti-CD3 (#RM-9107; Thermo Fisher Scientific; 1:300) or monoclonal mouse anti-Ki67 (#NCL-Ki67p; Novocastra, Leica Biosystems; 1:1000) antibodies, using standard protocols. Images were obtained using an Olympus BX 53 LED light microscope with an Olympus SC50 camera.

The mice were killed by carbon dioxide (CO₂) inhalation, and the tumors were removed and frozen in Tissue-Tek O.C.T. compound at −80°C. A standard streptavidin horseradish immunoperoxidase method was used for human CD3 staining as described previously [16 (link)]. Briefly, 7-μm cryosections were fixed in cold acetone for 5 minutes at 4°C and blocked with a peroxidase blocking system for 10 minutes (Dako, Carpinteria, CA, USA). Routine immunostaining was carried out with an initial 30-minute incubation with 10% normal goat serum at room temperature (RT), primary rabbit anti-human CD3 for T cells (RM-9107; Thermo Scientific, Waltham, MA, USA) at 1:100 dilution at RT for 45 minutes, secondary biotinylated goat anti-rabbit antibody at 1:200 dilution at RT for 30 minutes and streptavidin-biotinylated horseradish peroxidase complex reagent (Dako) at RT for 30 minutes, followed by washing three times for 5 minutes each after each incubation. Sections were then exposed to DAB + chromogen (Dako) for 5 minutes at RT, counterstained with hematoxylin, dehydrated, cleared and mounted.