Rabbit anti tyrosine hydroxylase

Immunofluorescence microscopy was performed on 35-μm brain slices containing the region of interest (see Additional file 1: Figure S1a). The following primary antibodies were used: rabbit-anti Iba-1 (1:500, Wako); rabbit anti-GFAP (1:250, Thermo Fisher); mouse monoclonal anti-NeuN (1:250, Millipore); goat anti-IL-13 (1:50, R&D system), rabbit anti-3-Nitrotyrosine (3-NT, 1:200, Millipore). After washing, a combination of secondary antibodies was used for detection: 488 Alexa goat anti-rabbit, 594 Alexa goat anti-mouse IgG1 (Invitrogen). Slides were counterstained with DAPI and were cover-slipped in Fluoromount. Digital images were taken using a Zeiss LSM 710 laser scanning confocal microscope (LSCM). Tyrosine Hydroxylase DAB Immunohistochemical analysis was performed using rabbit anti-Tyrosine Hydroxylase, (1:10000; Millipore), incubated with a biotinylated goat-anti rabbit antibody (1:400; Vector Labs) for 1 h at RT and then stained with Vectastain ABC Kit (Standard) and DAB Peroxidase Substrate (Vector Labs). For cresyl violet (CV) staining (Nissl), adjacent sections were mounted on positive charged slides and air-dried. The slides were soaked in CV solution (0.25% cresyl violet and 0.3% acetic acid) for 15 min (warmed at 50 °C), were dehydrated with alcohol and xylene, and were cover-slipped with Vectamount.

Brain fixation and antibody staining were carried out according to the standard protocol. Briefly, adult brains were dissected in phosphate buffered saline (PBS) then fixed with 4 % formaldehyde followed by a few washes in PBT (PBS + 0.1 % Triton X-100). After blocking with 3 % BSA (in PBT), brain samples were incubated in primary antibody (rabbit anti-tyrosine hydroxylase, Sigma, 1:1000 dilution) overnight at 4 °C with rotation. After removal of primary antibodies, samples were washed with PBT followed by incubation in secondary antibody (Cy3-conjugated goat anti-rabbit, Jackson Immunoresearch, 1:500). After washes, samples were incubated with Vectashield and analysed by Carl Zeiss Upright Confocal Microscope.

Flies were aged to Day 20 and Day 60 after eclosion before fly brains were dissected, fixed with paraformaldehyde, and stained according to published protocols [42 (link)]. Brains were probed with rabbit anti-tyrosine hydroxylase (1:500, Sigma-Aldrich #T2928). For the DA neuron count, the number of neurons in the five different clusters was scored under confocal microscopy and subjected to statistical analysis performed in Graph Pad Prism 6.

Protein was extracted from fly head homogenates and equal amounts of protein from the various genotypes were resolved by SDS-PAGE. HA-tagged VPS35 was detected using mouse anti-HA (1:1000, Santa Cruz). Flies were aged to day 20 and 60 after eclosion, before fly brains were dissected, fixed and stained according to published protocols [14 (link)]. Brains were probed with rabbit anti-tyrosine hydroxylase (1:500, Sigma-Aldrich) and mouse anti-HA (1:200, Santa Cruz).

Dulbecco's Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS) and other cell culture reagents, goat anti-mouse- AlexaFluor594 (H + L) antibody, goat anti-rabbit-AlexaFluor488 (H + L) antibody and rabbit IgG isotype control antibody were obtained from Thermo Fisher Scientific (Invitrogen). si-m-PYCARD/ASC RNAs were obtained from Guangzhou RiboBio Co., Ltd. Mouse anti-NLRP3 (Cat# AG-20B-0014), mouse anti-caspase-1 (Cat# AG-20B-0042) and rabbit anti-ASC (Cat# AG-25B-0006) were purchased from AdipoGen Life Sciences. Rabbit anti-α-synuclein (Cat# 4179), rabbit anti-Phospho-α-synuclein (Cat# 23706), rabbit anti-phospho-NF-κB p65 (Cat# 3033) and mouse anti-IL-1β (Cat# 12242) were purchased form Cell Signaling Technology. rabbit anti-ASC (Cat# YT0365) was purchased from ImmunoWay Biotechnology. Mouse anti-Phospho-α-synuclein (Cat# pSyn #64) and rabbit anti-IBA1 (Cat# 019-19741) were purchased from FUJIFILM Wako Pure Chemical Corporation. Mouse anti-α-synuclein (Cat# sc-12767) and mouse anti-GSDMD (Cat# sc-393581) was purchased from Santa Cruz Biotechnology. Rabbit anti-NF-κB p65 (Cat# ab16502), goat anti-IBA1 (Cat# ab5076) and rabbit anti-GSDMD (Cat# ab219800) were purchased from Abcam. Rabbit anti-IL18 (Cat# A1115) was purchased from ABclonal. Rabbit anti-tyrosine hydroxylase (Cat# AB152) was purchased from Merck Millipore.