Rabbit anti th

Primary rat microglial cells were seeded on poly-l-lysine-coated coverslips in 24-well plates and treated with CD200Fc, LPS and α-syn. Twenty-four hours later, the cells were fixed with 4% paraformaldehyde for 30 min, washed in PBS, and permeabilized with 0.3% Triton X-100 in PBS for 10 min at room temperature. After blocking with 10% normal horse serum for 1 h, the cells were incubated with goat anti-CD200R1 (1:100; Santa Cruz) overnight at 4°C. The next day, they were incubated with an Alexa Fluor 594-conjugated (1:500; Life Technologies) and/or an Alexa Fluor 488-conjugated secondary antibody (1:500; Life Technologies) for one hour at room temperature.
Brain tissue sections were prepared as previously described [26 (link)]. Briefly, nigral coronal sections (40 μm) from frozen paraformaldehyde-fixed brains were collected serially and stored at -20°C in tissue collection solution (50% 0.01 M PBS/50% glycerol). The sections were permeabilized in 0.01 M PBS containing 0.3% Triton X-100, blocked in 5% normal horse serum, and incubated overnight at 4°C with mouse anti-tyrosine hydroxylase (TH) (1:?2000, Sigma), rabbit anti-Iba-1 (1:500, Wako), rabbit anti-TH (1:1000, Novus) and mouse anti-human α-syn (1:500, Santa Cruz), followed by incubation with a secondary antibody as described above and imaged with a confocal microscope (Leica TCS SP8).

Mice were euthanized with CO₂ and hind limbs at the ankle were collected and were fixed in Zamboni fixative (Newcomers Supply, Middleton, WI) for 24–72 hours. Footpads were dissected out and were washed in phosphate buffer and placed in cryoprotectant (30% glycerol) solution. Tissue blocks were cut by freezing microtome at 50 μm intervals and immunohistochemical staining was performed using a standard chromogen technique with the following antibodies: rabbit anti-PGP 9.5 (AbD Serotec, a Bio-Rad Company, Kidlington, UK) and rabbit anti-TH (Novus Biologicals, Littleton, CO). For each marker, four 50 micron sections were selected from footpads 3 and 4. These were selected at random from throughout the possible sections. Sections were incubated over night at room temperature in 96 well tissue culture plates on a horizontal tabletop shaker at 50 rolls per minute. The following day, sections were washed in phosphate buffer 2–3 times and then incubated with biotinylated goat anti-rabbit Ab (Vector Labs, Burlingame, CA) for 2–3 hr. Bound immunoglobulin was visualized by the ABC kit (Vector labs, Burlinggame, CA).

For all imaging without staining, adult flies were anesthetized on ice and dissected in cold phosphate buffered saline (PBS). Brains or ventral nerve codes (VNCs) were fixed in 2% paraformaldehyde (weight/volume) for 30 min, washed with washing buffer (PBS with 1% Triton X-100, v/v, 3% NaCl, g/ml) for 7 min three times, and mounted in Focusclear (Cell Explorer Labs, FC-101).
For imaging with staining, brains and VNCs were fixed for 30 min and washed for 15 min three times. Then they were blocked in PBSTS, incubated with primary antibodies, washed with washing buffer, incubated with second antibodies, and mounted as described previously (Dai et al., 2021 (link); Dai et al., 2019 (link)).
All brains or VNCs were imaged on Zeiss LSM710 or Zeiss LSM880 confocal microscope and processed with Imaris.
The following primary antibodies were used: mouse anti-PDF (1:200, DSHB), rabbit anti-TH (1:1000, Novus Biologicals), and rabbit anti-LK (1:1000, Rao Lab, this paper). Rabbit anti-DSK (1:1000) was a gift from Dr. C. Zhou Lab (Institute of Zoology, Chinese Academy of Science) (Wu et al., 2020 (link)). The following secondary antibodies were used: Alexa Fluor goat anti-mouse 488 (1:1000, Invitrogen) and Alexa Flour goat anti-rabbit 488/633 (1:1000, Invitrogen).
For Figure 2, the number of TH-positive neurons was counted with Imaris Spots plugin.