Cd11b efluor 660

Tumor tissues were harvested from mice 2 days after the last injection, embedded in OCT (Leica) and cut into 8 µm cryosections on slides. Subsequently, samples were fixed with ice cold acetone for 15 min at −20 °C, and permeabilized for 5 min with 0.5% Triton X-100 in phosphate-buffered saline (PBS) (Ki-67 assay) or not. Blocking was achieved using 10% horse serum and 1% BSA in PBS for 1 h at room temperature. Tissues were stained at 4 °C overnight using the appropriate antibodies diluted in blocking solution. Antibodies used for immunohistochemistry were purchased from Biolegend (CD8a PE, cat#100708; NK 1.1 Alexa Fluor^® 647, cat#108720; Ly-6G/Ly-6C (Gr-1) Alexa Fluor^® 488, cat# 108417, and their isotype control) and eBioscience (Ki-67 FITC, cat#652410; CD31 FITC, cat#11-0311-81; CD11b eFluor 660, cat#50-0112-80, and their isotype control). Apoptotic cells in the tumor tissue sections were detected using a TUNEL assay kit (cat#C1089, Beyotime), and the staining was performed according to the manufacturer’s recommendations. Nuclear counterstaining was performed using DAPI. Slides were mounted with ProLong gold antifade mountant (cat#P36930, Thermo Fisher Scientific). Images were captured using Olympus BX51 upright microscope.

Cells cultured in vitro or isolated from tumors or spleens were processed for surface labeling with appropriate antibodies. Propidium iodide was used to distinguish the live cells. Cells were further permeabilized using 70% ethanol or True-Nuclear™ transcription factor buffer set (cat# 424401, BioLegend) and stained for Ki-67 or FOXP3. Data were acquired using the Beckman CytoFLEX flow cytometer and analyzed using FlowJo software. Antibodies used for flow cytometry were purchased from Biolegend (anti-mouse CD16/32, cat#101320; CD8a Alexa Fluor^® 647, cat#100724; NK 1.1 Alexa Fluor^® 647, cat#108720; Ly-6G/Ly-6C (Gr-1) Alexa Fluor^® 488, cat#108417; CD19 FITC, cat#152404; CD45R/B220 Alexa Fluor^® 647, cat#103226; F4/80 Alexa Fluor^® 488, cat#123120; CD206 Alexa Fluor^® 647, cat#141712; FOXP3 Alexa Fluor^® 488, cat#126406; CD4 Alexa Fluor^® 647, cat#100530; CD11c Alexa Fluor^® 488, cat#117311; H-2K^b/H-2D^b FITC, cat#114606; I-A/I-E Alexa Fluor^® 647, cat#107618; CD80 Alexa Fluor^® 647, cat#104718; CD86 FITC, cat#105006; CSF-1R Alexa Fluor^® 488, cat#135511) and eBioscience (anti-mouse Ki-67 FITC, cat#11-5698-82; CD3ε FITC, cat#11-0031-82; CD11b eFluor 660, cat#50-0112-82).