Af6000 fluorescent microscope

Fibroblasts of patient (P2) and healthy donor were stimulated with 100 ng ml⁻¹ of lymphotoxin α1/β2 (R&D Systems, 678-LY-010) for 4 h. After stimulation, cells were fixed with 4% formaldehyde in PBS for 30 min and then blocked and permeabilized with solution containing 10% FCS plus 0.1% Triton X-100. Cells were immunostained with DAPI and rabbit antibodies against NFκB2 (p100/p52) (Cell Signaling, 3017) and NF-κB (p105/p50) (Cell Signaling, 3035) at a dilution of 1:100, respectively, and afterwards with anti-rabbit Alexa Fluor 546-conjugated antibody at a dilution of 1:500 (Life Technologies, A10040). Images were acquired on a Leica AF6000 fluorescent microscope using Leica LASAF software for acquisition. Images were taken at × 64 magnification.

Transgenic cell lines cultured on PDL-coated coverslips were fixed in paraformaldehyde 4% for 20 min at RT and rinsed in PBS 3 times. Samples were permeabilized and blocked with PBS containing 0.1% Triton, 1% BSA and 5% normal goat serum for 60 min at RT.
Samples were incubated with rabbit anti MAP2 (1:1000, Abcam), mouse anti α-synuclein (1:500, Invitrogen) or rabbit anti TOM20 (1:1000 Santa Cruz FL-145) antibodies at RT for 1 h followed by incubation of fluorescent secondary antibodies (1:400) and Hoechst for 1 h at RT. Images were acquired using a Leica AF-6000 fluorescent microscope.