Alexa fluor 488 anti mouse cd4 antibody

Draining LNs were isolated from graft recipients on postoperative day 42, and a single-cell suspension was passed via a 70 μm cell strainer (Corning, CA, USA). Viable single cells were subjected to plate counting as 5 × 10⁵ cells/well in 96-well plates on RPMI media (Welgene Inc., Gyeongsan-si, Republic of Korea) with 1% fetal bovine serum (FBS; Gibco BRL, Karlsruhe, Germany) for 48 h. The cultured single cells were harvested and immunostained with the following antibodies: PE-anti-mouse CD11c antibody (1:100, host:hamster, cat# 117307, BioLegend, San Diego, CA, USA), Alexa Fluor 647-anti-mouse I-Ad antibody (1:100, host:mouse, cat# 115010, BioLegend, San Diego, CA, USA), Alexa Fluor 488-anti-mouse CD4 antibody (1:100, host:mouse, cat# 100423, BioLegend, San Diego, CA, USA), and PE-anti-mouse IFN-gamma antibody (1:100, host:rat, cat# 12-7311-82, Invitrogen, Carlsbad, CA, USA). All antibodies were stained appropriately with the matched isotype controls. The stained cells were analyzed with FACS Melody (BD Biosciences, Franklin Lakes, NJ, USA) and FlowJo software X 10.5.3 (FlowJo LLC, Ashland, OR, USA).

Tumours were harvested from the mice and frozen in the cutting medium before sectioning via a cryotome. Before staining, the tissues were incubated with 1% BSA to block the nonspecific binding. For blood vessel staining, 10 μg mL⁻¹ of anti-mouse CD31 (R&D Systems, catalogue no. AF3628, goat IgG) antibody was incubated with the slides overnight, and the donkey anti-goat IgG (Invitrogen, catalogue no. 2044862, AF568-conjugated) secondary antibody was added after three times PBS washing, the nuclei were stained with Hoechst 33342. For platelet staining, 10 μg mL⁻¹ of anti-mouse CD62P (R&D Systems, catalogue no. AF737, goat IgG) were used. For the T cells staining, 20 μg mL⁻¹ PE anti-mouse CD8a antibody (Biolegend, cat no. 100708, clone: 53-6.7) and Alexa Fluor® 488 anti-mouse CD4 antibody (Biolegend, cat no. 100425, clone: GK1.5) was used to stain the tumour slices overnight at 4 °C. The tumour cell apoptosis was analysed with TUNEL. The stained slides were measured on a Zeiss LSM880 confocal laser scanning microscope and analysed with Zen 2 and Image J software.