Formalin-fixed paraffin-embedded tissues were used for histology and immunohistochemistry. Two pathologists, blinded to experimental group, evaluated sections for pathology and inflammation. Inflammation was scored on a 0–4 scale (0, normal mucosa; 1, minimal inflammation (occasional scattered granulocytes and leukocytes); 2, mild inflammation (scattered granulocytes with occasional infiltrates); 3, moderate inflammation (scattered granulocytes with patchy infiltrates); and 4, severe inflammation (multiple extensive areas with abundant granulocytes and marked infiltrates), as previously described (9 (link)).
Immunohistochemistry was performed in EDTA antigen retrieval buffer, as previously described (6 ), using antibodies against CD11b (Abcam), F4/80 (Abcam), and Ly6G (Novus Biologicals). DAB-positive cells in tumor-positive fields of view were acquired at 10X magnification and quantified using Viziopharm acquisition and analysis software (Hoersholm, Denmark), and expressed as the number of positive cells in each histologically defined area. Single cell suspensions were generated from spleen and cervical lymph nodes from tumor-bearing mice for flow cytometry. Cells were stained according to manufacturer’s protocol against CD11b-APC, Ly6C-FITC, Ly6G-Pacific Blue, and F4/80-PE with at least 10,000 events analyzed by Miltenyi MACSQuant cytometer and software.
Immunohistochemistry was performed in EDTA antigen retrieval buffer, as previously described (6 ), using antibodies against CD11b (Abcam), F4/80 (Abcam), and Ly6G (Novus Biologicals). DAB-positive cells in tumor-positive fields of view were acquired at 10X magnification and quantified using Viziopharm acquisition and analysis software (Hoersholm, Denmark), and expressed as the number of positive cells in each histologically defined area. Single cell suspensions were generated from spleen and cervical lymph nodes from tumor-bearing mice for flow cytometry. Cells were stained according to manufacturer’s protocol against CD11b-APC, Ly6C-FITC, Ly6G-Pacific Blue, and F4/80-PE with at least 10,000 events analyzed by Miltenyi MACSQuant cytometer and software.
