Primary anti gfp

LECs sorted from LNs of LC3-GFP mice were starved (HBSS, devoid of FBS) and treated with CQ (50 µM) for 4 h to induce and stabilize autophagosomes. Then, LECs were fixed in PFA 4% at RT for 20 min. After washing, samples were permeabilized with Triton X-100 (1/400e) and blocked with normal goat serum (1/100e) for 30 min at RT and with Triton X-100 (1/800e) for another 30 min at RT. Staining was performed overnight at 4°C using primary anti-GFP (1/1,000e; Thermo Fisher Scientific) and anti-SphK1 or anti-SphK2 (1/200e; Bioss USA) antibodies. After washing, secondary antibodies against chicken Alexa Fluor 488 for GFP staining and anti-rabbit Alexa Fluor 546 (1/1,000e; Thermo Fisher Scientific) were incubated for 2 h at RT (in the dark).

LC3-GFP mice were injected s.c. with IFN-γ (1 µg/ml) and with CQ 24 h later (50 µM) in both flanks and neck area or immunized for CIA. Stromal cells (LECs, BECs, and FRCs) from dLNs were sorted (Astrios; Beckman Coulter) after 24 h. Sorted cells were cytospined and fixed in acetone at −20°C for 20 min. After washing, samples were permeabilized with Triton X-100 (1/400e) and blocked with normal goat serum (1/100e) for 30 min at RT and Triton X-100 (1/800e) for another 30 min at RT. Staining was performed overnight at 4°C using primary anti-GFP (1/1,000e; Thermo Fisher Scientific) and anti–MHC-II biotinylated (1/200e) in some experiments. After washing, secondary antibodies against chicken Alexa Fluor 488 for GFP staining and streptavidin Alexa Fluor 546 for MHCII detection (1/300e; eBioscience) were incubated for 2 h at RT (in the dark).