Neuroscience Antibodies

…for 1 hour at room temperature to block nonspecific binding sites and then incubated in primary antibody (rabbit anti- tyrosine hydroxylase, Pel Freez Biologicals #P40101-150, 1:250 in blocking solution) for 72 hours at 4oC on a shaker. Sections were washed in between incubation steps with 6 x 3min PBS-Tx. Sections were incubated in diluted secondary antibody (biotinylated donkey anti-rabbit Ig

…1 h at ambient temperature. As for the immunoblots, the antibodies against parkin C-Terminal region (monoclonal parkin clone [PRK8], 1:1,000 and polyclonal parkin (cell signaling cat#2132, 1:1,000)), TH (Pel-Freez Biolo- gicalsCat# P40101-150, 1:1,000),αsyn(SYN1,BDBiosciencesCat# 610787, 1:1,000), MJFR1 (Abcam Cat# ab138501 [MJFR1], 1:1,000) and αsyn phosphorylatedatserine129(αsynP129)(AbcamCat#ab168381[MJF-R13…

…times for 5 min each using 1× PBST. Samples were placed in blocking buffer (1% bovine serum albumen in PBST) for 1 h at room temperature and then incubated with anti-Tyrosine Hydroxylase (1:500, P40101 PelFreez) for 3 days at 4 °C. After the primary was removed, samples were washed 3 times with 1× PBST for 5 min each, then incubated in secondary (1:500 Alexa Fluor anti-Rabbit 555 (A-21428) and 1:1000 DAPI) for 1…

…an investigator blinded to the genotype. ARTICLE IN PRESS ARTICLE IN PRESS Antibodies and Reagents The antibodies and reagents used in the study include: Anti-Tyrosine Hydroxylase (rabbit, 1:5000 for IF, Pel-Freez, P40101-0), Anti-Tyrosine Hydroxylase (mouse, 1:500 for IF, Millipore, MAB5280), Anti-Kinesin 5B heavy chain (mouse, 1:500 for WB, Millipore, MAB1614), Anti- Dynein medium chain (mouse, 1:1000 for WB,…

…Peprotech, 450-10-50UG). Neuronal cultures were assessed by immunostaining with antibodies against Mikrotubuli-associated protein 2 (MAP2; Abcam ab5392, dilution 1:10000) and Tyrosin Hydroxylase (TH; Pel-Freez P40101, dilution 1:1000). 2.2 Live cell imaging of mitochondrial calcium Neurons were cultivated in 8 well chamber slides (Ibidi, 80806) at 100,000 cells per well. Then, neurons were transfected with 0.5…

…then incubated at 4 °C overnight with a rabbit polyclonal primary antibody against TH, GFAP or IBA-1 in KPBS containing 0.4% triton X-100 (TH, Pel Freez #P40101- 150, 1:1000; GFAP, Proteintech #16,825–1-AP 1:500; and Iba-1, Novus biologicals Cat#NBP2-19,019, 1:500). After five to six rinses in KPBS, the sections were treated for two hours in a biotinylated secondary antibody (PK- 6200, 1:1000,