Immunohistochemical and microscopy evaluation of frozen tumor sections. (A) Immunohistochemical evaluation of frozen tumor sections. The micrograph shows the following: high number of macrophages (brown cytoplasm staining to monoclonal CD68 antibody) infiltrating neoplastic tissue; monoclonal antibody to mouse Dectin-1 identifies a high number of small immunopositive cells (cells with brown cytoplasm) intermingled with neoplastic elements; immunohistochemistry with monoclonal antibodies to mouse interleukin-10, mMGL1, CXCL16, and NOS2 identifies isolated positive elements (brown cells); immunohistochemistry with biotinylated anti-mouse CRG-2/IP10 antibody (CXCL10) shows a cluster of positive small cells, whereas isolated elements are not observed; immunohistochemistry to CCL2, CCL5, and CXCL9 shows similar patterns with clusters of positive brown cells and few isolated positive cells. Original magnifications: × 20 (CD68, Dectin-1, CXCL10), × 40 (IL-10, mMGL1, NOS2, CCL2, CCL5, CXCL9, CXCL16). (B) Laser scanning confocal microscopy evaluation of frozen tumor sections: double immunostaining with monoclonal antibodies to mouse CD68 (red) and Dectin-1, IL-10, CRG2/IP10/CXCL10, mMGL1, NOS2, CCL2, CCL5, CXCL9, CXCL16 (all green staining). As shown in the micrographs, Dectin-1, IL-10, CRG-2/IP10, mMGL1, CCL2, CCL5, CXCL9, CXCL16 are expressed in macrophages but not in the totality of the CD68⁺ elements (red staining); NOS2-CD68 colocalization is observed in very rare cells.