Rabbit anti rab7 h 50

Duolink™ (OLINK Bioscience) was performed according to the manufacturer’s instructions. Briefly, the cells were incubated for 30 min on IBItreat μ-channels (IBIDI) coated with poly-L-Lysine, fixed for 10 min in 4% paraformaldehyde prewarmed at 37 °C, permeabilized in PBS, 0.2% saponin for 10 min and blocked with 0.2% BSA in PBS, 0.1% saponin. Primary antibodies used were: mouse anti-CD3ε (OKT3, Biolegend), mouse anti-CD3ζ (6B.10.2, Santa Cruz Biotechnology), rabbit anti-IRAP (a generous gift from S. Keller, Virginia University, USA), mouse anti-Lck (3A5, Santa Cruz Biotechnology), rabbit anti-LAT (Millipore), rabbit anti-Rab7 (H-50, Santa Cruz Biotechnology) and rabbit anti-IRAP (D7C5), mouse anti-IRAP (3E1) and rabbit anti-ZAP-70 (D1C10E) (all from Cell Signaling Technology). All primary antibodies were diluted at 1/100, except mouse anti-CD3ζ (1/50). After washing the cells, PLA probes were added, followed by hybridisation, ligation, and amplification for 100 min at 37 °C. Protein interactions were visualised after incubation with the detection solution. Fluorescence signal was acquired on an LSM 510 Zeiss confocal microscope.

The rat monoclonal antibody anti-LAMP1 (clone ID4B) was obtained from Beckton Dickinson, the rabbit anti-Rab7 (H-50) was from Santa Cruz Biotechnology, the mouse anti-H-2K^b clone Y3 was a gift from Dr. Gunter J. Hammerling, German Cancer Research Center, Heidelberg, Germany. Armenian hamster anti-CD80 (clone 16-10A1) conjugated to PE, rat anti-CD86 (clone GL1) conjugated to FITC, rat anti-MHC-II (I-A/I-E, clone M5/114), and mouse anti-H-2Kb/pOVA_257-264 (SIINFEKL) conjugated to PE were from Thermo Scientific. AF-488-conjugated chicken anti-mouse and AF-488-conjugated goat anti-rat (Invitrogen) were used as secondary antibodies