WB
Western blot analysis of Dopamine D2 Receptor on different lysates with Rabbit anti-Dopamine D2 Receptor antibody at 1/10,000 dilution.
Lane 1: SH-SY5Y cell lysate (no heat),
Lane 2: U-87 MG cell lysate (no heat),
Lane 3: NCI-H1299 cell lysate (no heat),
Lane 4: THP-1 cell lysate (no heat),
Lane 5: Neuro-2a cell lysate (no heat),
Notice: no heat means the lysate is not boiled.
Lysates/proteins at 20 µg/Lane.
Exposure time: 10 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 1/10,000 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.
IICC/IF
Immunocytochemistry analysis of SH-SY5Y cells labeling Dopamine D2 Receptor with Rabbit anti-Dopamine D2 Receptor antibody at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Dopamine D2 Receptor antibody at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (488) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (594) was used as the secondary antibody at 1/1,000 dilution.
IF-F
Immunofluorescence analysis of frozen mouse striatum tissue with Rabbit anti-Dopamine D2 Receptor antibody at 1/500 dilution.
The section was not undergone antigen retrieval. The tissues were blocked in 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (green) at 1/500 dilution overnight at 4 ℃, washed with PBS. Goat Anti-Rabbit IgG H&L (488) was used as the secondary antibody at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).
FC
Flow cytometric analysis of Neuro-2a cells labeling Dopamine D2 Receptor.
Cells were fixed and permeabilized. Then stained with the primary antibody (1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).