Related topics: Alphabetical list, Neurodegeneration, Neurotransmission
On October 1, 2019
Trine Rasmussen wrote:
This antibody is also good for fluorescence ICC staining of Ser129 phosphorylated α-synuclein in cells on coverslips.
100.000 P0 mice hippocampal neurons were seeded pr. laminin/poly-D coated coverslip. At 5DIV neurons were treated with 100 nM α-synuclein preformed fibrils (StressMarq, SPR-324) in 0.5 ml fresh medium with supplements. The fibrils were sonicated in a water bath for 1 hour immediately prior to use. 0.5 ml new medium was added instead of the usual 50% media change ( e.g. 8DIV).
At 10DIV cells were fixed with 4% PFA for 20 minutes at room temperature. Followed by:
• 3×10 min. wash in PBS.
• 3×10 min. wash in PBS with 0.1 % Triton X-100.
• 30 min. blocking in 0.5 mL PBS with 10 % FBS.
• Incubation over night at 4°C with primary antibodies diluted in PBS with 10 % FBS
• 3×5 min. wash in PBS with 0.1 % Triton X-100
• 4 hours of incubation at RT with secondary antibody in PBS with 10 % FBS.
• 2×5 min wash in 0.5 mL PBS
• 5 min wash in 0.5 mL PBS with Hoechst nuclear stain
The cover slips were mounted, and edges were sealed with nail polish.
The neurons were visualized with a confocal microscope and Ser129 phosphorylated α-synuclein was clearly visible perinuclearly and in neurites compared to untreated controls.
On August 15, 2019
Trine Rasmussen wrote:
This antibody is very good for fluorescence IHC staining of α synuclein on free floating brain sections.
Mice were injected with α-synuclein AAV vector in the right hemisphere (striatum). The left hemisphere was left untreated. After 4 months the mice were sacrificed. Their brains were removed and fixed in PFA and cut into 20 µm sections. The tissue was stained using a free floating immunohistochemistry protocol. Clear streaks of stained α-synuclein were visible at the site of injection compared to control.
Sections were collected in a multi-well plate with 300 µl 1x target retrieval solution (Dako, #S1699), incubated at 70 °C for 30 minutes and allowed cool at room temperature.
The sections were rinsed for 2 minutes in water, 3×10 minutes in TBS and 30 minutes in 1%BSA/0.3% Triton X/TBS and allowed to incubate with the 1:500 α synuclein antibody in TB buffer (50 mM) overnight at 4 °C with gentle agitation.
The staining was left at room temperature for 1 hour and subsequently rinsed 3×10 minutes in TBS. Sections were incubated with 1:300 secondary antibody in TB (50 nM) for 4 hours at room temperature. The sections were rinsed 3×10 minutes in TBS with hoechst added the last 10 minutes.
Sections were mounted on slides and allowed to dry.
Pictures were obtained using confocal microscopy.