Rabbit anti cd4

For ex vivo infiltrate immunofluorescence analysis, mice were injected according to the therapy schedule and euthanized 24 h after the last injection. Tumors were excised and embedded in a cryo-embedding medium (ThermoScientific, Waltham, MA, USA) and the corresponding cryostat tissue sections (8–10 μm thickness) were stained using the following primary antibodies: goat anti-CD31 (R&D Systems; AF3628), rabbit anti-Foxp3 (Invitrogen, 5H10L18; 7000914), rabbi anti-NCR1 (Abcam; ab214468), rabbit anti-CD4 (Sino Biological, Wayne, PA; 50134-R001), rabbit anti-CD8 (Sino Biological; 50389-R208) and rabbit anti-mouse CD274 (BiossUSA, Boston, MA, USA; bs1103R). Primary antibodies were detected with Donkey anti-rabbit AlexaFluor488 (Invitrogen; A11008) and Donkey anti-goat AlexaFluor594 (Invitrogen; A21209). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen; D1306). Slides were mounted with a fluorescent mounting medium (Dako Agilent, Carpinteria, CA, USA) and analyzed with a wide-field Leica TIRF microscope using the Leica LAS X Life Science Microscope Software. Quantification of tumor-infiltrating cells was made using Image J software [41 (link)].

For immunofluorescence analysis, tumors were embedded in a cryo-embedding medium (NEG-50, Thermo Fisher). Cryostat sections (8–10 μm) were stained using the following primary antibodies: goat anti-CD31 (1:200, R&D Systems; AF3628), rabbit anti-CD4 (1:200, Sino Biological, Wayne, PA; 50,134-R001), rabbit anti-Foxp3 (1:200, Invitrogen; 7000914), rabbit anti-NKp46 (1:200, BioLegend; 137602), and rabbit anti-CD8 (1:200, Sino Biological; 50389-R208). Primary antibodies were detected with anti-rabbit AlexaFluor488 (1:200, Invitrogen; A11008) and anti-goat AlexaFluor594 (1:200, Invitrogen; A21209). Cell nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI) (1:500, Invitrogen; D1306). Slides were mounted with mounting medium (Dako Agilent, Carpinteria, CA) and images were obtained with a Leica DMI6000B confocal microscope (Scientific Center for Optical and Electron Microscopy ScopeM, ETH, Switzerland). Images were analyzed using ImageJ software.