4.7. Immunofluorescence (IF) and Quantification

Immunofluorescence analyses were performed using the formalin-fixed paraffin-embedded 4-μm sections that were deparaffinized, fixed and boiled in 10 mM citric acid (pH 6.0) for 16 min at 600 W. Afterwards, cells were permeabilized and blocked with 0.1% Triton X-100, 4% NGS and 3% BSA solution and incubated with anti-Cc3 antibody (9664s, Cell Signaling Technology, Danvers, MA, USA, dilution: 1:1000) overnight at 4 °C and alexa 555 (A-21429, Thermo Fisher Scientific, Waltham, MA, USA, dilution 1:1000) for 1h. Between each step, tissues were washed three times with TBS-T, pH 8. Lastly, tissues were stained with Hoechst 33258 (dilution 1:2000) for nuclei staining and mounted using ProLong Glass Antifade Mountant ({"type":"entrez-protein","attrs":{"text":"P36984","term_id":"19858950","term_text":"P36984"}}P36984, Thermo Fisher Scientific, Waltham, MA USA). Images were obtained using a laser scanning confocal microscope (Olympus FluoView 1200; Olympus Corporation) equipped with an Olympus UPlanSAPO × 60 1.35 and an UPlanSAPO ×40 1.25 solid immersion lens oil immersion objective (Olympus). Cc3-positive cells were counted in 3 independent and representative high Power Fields (HPF) for each tumor sample under the confocal microscope (n = 2/treatment group/sex).