Rabbit igg isotype control antibody

Affinity purified rabbit anti-human LDL receptor IgG antibody was purchased from RDI Division of Fitzgerald Industries Intl, Concord, MA, USA and stored at −20 °C. Rabbit IgG isotype control antibody was purchased from abcam, Cambridge, UK and stored at +4 °C.

The GL261 cells were seeded in 8-well Permanox chamber slides (10,000 cells/well) and allowed to adhere. IFNγ (Recombinant Murine IFNγ, PeproTech) at a concentration of 50 ng/mL was added to the appropriate chambers after seeding. After 24 h, the chambers were removed and the cells were fixed with ethanol (96% v/v) for 15 min. The cells were permeabilized with 0.5% Triton-X 100 (Sigma-Aldrich) in PBS for 5 min and blocked with blocking solution (PBS, 0.25% Triton-X 100) containing 10% goat serum for 30 min at RT. Subsequently, the samples were incubated with the following primary antibodies overnight at 4 °C: PD-L1 (rabbit anti-mouse PD-L1, Abcam, Cambridge, UK) at a dilution of 1:50 (in PBS, 2% goat serum, 0.25% Triton-X 100) or Rabbit IgG Isotype Control Antibody (Abcam) at a solution of 1:170. The slides were washed three times with PBS and the cells were incubated with goat anti-rabbit AlexaFluor568 antibody (Invitrogen, ThermoFisher) at a solution of 1:1000 (in PBS, 2% goat serum, 0.25% Triton-X 100) for 1 h at RT followed by washing with PBS and nuclear staining with 4′, 6-diamidino-2-phenylindole (DAPI, 1:10,000 in distilled water). Slides were covered using Mowiol^®4-88/DABCO (CarlRoth, Karlsruhe, Germany). Fluorescence microscopy was performed using an Axiolab microscope (Zeiss) and ZEN lite 2.1, Zeiss, software.