Bcl2 124

Paraffin-embedded sections of each sample were immunostained. Antibodies (clones) used for immunohistochemistry were CD10 (56C6; Leica Microsystems, Wetzlar, Germany), CD20 (L-26; DakoCytomation, Glostrup, Denmark), BCL2 (124; DakoCytomation), BCL6 (P1F6; Leica Microsystems) and MUM1 (MUM1p; DakoCytomation). Each cases were considered positive if more than approximately 30% of the neoplastic cells were positive.

Immediately after mice sacrifice, tumors were fixed in 4% buffered formalin and paraffin embedded. Immunoreactions were revealed by Bond Polymer Refine Detection in an automated autostainer (BondTM Max, Leica Biosystems, Milan, Italy) using anti-F4/80 (SP115, ThermoFisher), -CD206 (Abcam, Cambridge, UK) or -bcl-2 (124, Dako, Milan, Italy) antibodies. For each tumor, three different 2 µm paraffin sections were analyzed and examined by light microscopy. The presence of F4/80 or CD206 positive cells was classified as peritumoral (PT) or intratumoral (IT) depending on their localization in the stroma surrounding the tumor islands or within the tumor-cell nests, respectively. IT-positive or PT-positive cells were counted in four high-power fields (HPF) per section and averages of positive cells/HPF in both compartments were calculated. Tumors were classified into four categories depending on the average number of cells/HPF (score 0: absence of positive cells; score 1: presence of <5 positive cells/HPF; score 2: presence of ≥5≤10 positive cells/HPF; score 3: presence of >10 positive cells/HPF). Evaluation of the IHC results was performed independently and in blinded manner by two investigators at 200× and 400× magnifications.