Vectashield medium

RAW264.7 cells were plated on Fn-coated cover for slides 12 hours before assay, starved, stimulated (0, 20, 60, and 120 min, 37°C) with LPS 1 μg/mL, and then washed by PBS three times and fixed with 4% paraformaldehyde (20 minutes, 20°C) in PBS. After being washed three times by PBS, cells were then permeabilized with 0.3% Triton X-100 (20 minutes, 20°C) and incubated (1 hour, 4°C) with 5% goat serum. Cells were cultured with a PTEN antibody and a TLR4 antibody overnight (4°C) and then washed by PBS three times, followed by fluorescence conjugated secondary antibody, respectively, for 1 hour (20°C) in a dark environment. After being washed by PBS three times, samples were mounted in Vectashield medium and images were recorded in a confocal microscope (Leica).

Brain sections were blocked in 1% bovine serum albumen (BSA) with 0.2% Triton X-100, before being incubated overnight at 4 °C with mouse anti-CHOP diluted in 1% BSA with 0.2% Triton X-100 in PBS. Sections were washed in PBS before being incubated with secondary antibody. Sections were washed in PBS, then mounted with Vectashield medium containing DAPI (Vector Laboratories, Burlingame, CA, USA).
Astrocytes and neurons isolated from LRRK2-WT and LRRK2-GS mice were treated with tunicamycin (0.2 μg/ml) and then fixed with 4% paraformaldehyde and then permeabilized with 0.25% triton for 10 min. Cells were washed three times in PBS and blocked with 1% BSA for 1 h. Cells were incubated with primary antibodies overnight at 4 °C. Washed cells were incubated with fluorescent secondary antibody (Invitrogen) for 1 h at room temperature. Cover slips were mounted with Vectashield medium containing DAPI and viewed on confocal microscope confocal microscope (TCS, DMi8; Leica, Germany).