Frozen tumor fragments were sectioned into 5 μm sections. Cryo-sections were fixed in a mix of acetone and methanol (2:1 volumes) at room temperature for 5 min. Air-dried sections were hydrated by three PBS incubations, 2 min each, at room temperature. Endogenous peroxidase activity was quenched with 3% H2O2 for 10 min, and endogenous biotin was blocked with the Avidin/Biotin Blocking Kit (Vector Laboratories, Burlingame, CA). We also performed a blocking step with 5% FBS and 1 μg/ml FcBlock (BD Biosciences) for 30 min in PBS, prior to incubation of sections with primary antibody. Rat anti-CD45 antibody was diluted in 5% FBS in PBS and incubated on the tissue for 1 h. Sections were washed and the antibody was detected using the ABC Vectastain kit and DAB, 3,3′-diaminobenzidine (Vector Laboratories, Burlingame, CA). We performed counterstaining with Harrys Hematoxylin (VectorLaboratories) and Eosin (Sigma–Aldrich), dehydrated the tissue sections and mounted with Permount (Thermo Fisher Scientific, Waltham, MA) and a coverslip. Images were acquired with a BX61 Olympus microscope (Olympus Corporation, Tokyo, Japan).
Transversal 5 μm spleen cryo-sections, were fixed as described above, stained with Harrys Hematoxylin, washed, dehydrated, and mounted with Permount and a coverslip. Images were acquired with a BX61 Olympus microscope (Olympus Corporation).
Transversal 5 μm spleen cryo-sections, were fixed as described above, stained with Harrys Hematoxylin, washed, dehydrated, and mounted with Permount and a coverslip. Images were acquired with a BX61 Olympus microscope (Olympus Corporation).