Related topics: Alphabetical list, Neurodegeneration, Neuroscience, Neurotransmission
On October 7, 2019
Trine Rasmussen wrote:
I was not able to detect Ser129 phosphorylated α-synuclein on free floating brain sections using this antibody with our usual fluorescence IHC staining protocol. Instead I recommend SPC-742, also from STRESSMARQ.
Following protocol was used on brain slides from mice injected with α-synuclein AAV vector in the right hemisphere (striatum) (the left hemisphere was left untreated).
4 months post injection the mice were sacrificed, and their brains were fixed in PFA and cut into 20 µm sections. 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 1:500 or 1:200 dilution 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.