ICC/IF
ICC staining of TMEM119 in SH-SY5Y cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 10% negative goat serum for 15 minutes at room temperature. Cells were probed with the primary antibody (1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 conjugate-Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
WB
Western blot analysis of TMEM119 on different lysates with Rabbit anti-TMEM119 antibody at 1/5,000 dilution.
Lane 1: Mouse brain tissue lysate
Lane 2: Mouse hippocampus tissue lysate
Lysates/proteins at 20 µg/Lane.
Predicted band size: 29 kDa
Observed band size: 45 kDa
Exposure time: 24 seconds;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50,000 dilution was used for 1 hour at room temperature.
FC
Flow cytometric analysis of TMEM119 was done on SH-SY5Y cells. The cells were fixed, permeabilized and stained with the primary antibody (1ug/ml) (red) compared with Rabbit IgG, monoclonal - Isotype Control (green). After incubation of the primary antibody at +4℃ for 1 hour, the cells were stained with a Alexa Fluor®488 conjugate-Goat anti-Rabbit IgG Secondary antibody at 1/1,000 dilution for 30 minutes at +4℃ (dark incubation).Unlabelled sample was used as a control (cells without incubation with primary antibody; black).