Ab6703

CT26^hEGFR tumors from different treatment groups were collected after 13 days of implantation and fixed in 10% neutral buffered formalin (Sigma-Aldrich) for 48 h. Then, after extensive washing in PBS, tissues were embedded in paraffin, cut at 3 µm, mounted in Superfrost®plus slides, and dried overnight. Slides were deparaffined in xylene and re-hydrated through a series of graded ethanol washes, ending in a final rinse in pure water. Slides were incubated with a rat monoclonal anti-CD8a (1:200 dilution) (OTO94A; Monoclonal Antibodies Core Unit CNIO) followed by a rabbit anti-rat secondary antibody (cat#ab6703, Abcam) and a visualization system (Novolink Polymer anti-Rabbit, Leica) conjugated with HRP. Nuclei were counterstained with Harris’ hematoxylin. Positive control sections known to be primary antibody positive were included for each staining run. Whole digital slides were acquired with a slide scanner (AxioScan Z1, Zeiss), and total versus positive cells were automatically quantified (AxioVision 4.6 software package, Zeiss).

The spleen of infected and non-infected mice was collected and frozen in tissue-Tek O.C.T freezing medium at −80°C until analysis. Tissues were sectioned at 6 μm, mounted on Superfrost+ glass slides (Fischer Scientific). Splenic sections were fixed in cold solution (−20°C) of 50% acetone (VWR, 20065.293) and 50% methanol (Millipore, 1.06009.5000) for 5 min, then incubated with protein serum free (DAKO, X0909) for 30 min at RT. The sections were then incubated overnight in dark wet room with 100 μl of an anti-macrophage scavenger receptor (MARCO) mAb (ED31, T-2026, BMA Biomedicals, Switzerland) solution (2 μl/mL), followed by a 30 min incubation at RT with 100 μl of a polyclonal rabbit anti-rat Immunoglobulins (ab 6703, abcam, UK) solution (4 μg/mL). Positive staining were revealed using goat anti-rabbit IgG (H+L) Alexa fluor 488 conjugate (A11034, Invitrogen, Belgium), incubated for 2 h at RT. All antibodies were diluted by DAKO real antibody diluent (S2022, DAKO). Slides were washed 3 times for 5 min after each step by distilled water, mounted with ProLong® Gold Antifade Mountant (P10144, Thermofisher, Belgium), viewed and photographed with an Olympus FSX100 inverted microscope.