Cy3 conjugated goat anti rabbit

The adult Drosophila brains were dissected in PBS, fixed (4% formaldehyde), washed (PBT: PBS + 0.1% Triton X-100), blocked (3% BSA in PBT) for 1 h at room temperature/RT and then incubated in the primary antibody (rabbit anti-Tyrosine Hydroxylase/TH/Sigma, 1:1000 dilution) overnight at 4 °C. On the next day, samples were washed and incubated in the secondary antibody (Cy3-conjugated goat anti-rabbit/Jackson Immunoresearch, 1:500 dilution) for 1.5 h at RT, washed and added the Vectashield mounting media (Vector Laboratories). Stained samples were analyzed using Leica TCS SP8 inverted microscope. Images were collected as z-stacks, each with the size of 1 µm.

The following primary antibodies were used: mouse anti-α-Syn (BD610787, BD Biosciences, Franklin Lakes, NJ), rabbit anti-V5 (ab9116, Abcam, Cambridge, UK), mouse anti-α-Syn (LB509, Covance, Munich, Germany), rabbit anti-TH (Zytomed, Berlin, Germany), rabbit anti-TPH2 (ABN60, Millipore, Darmstadt, Germany), mouse anti-NeuN (MAB377, Millipore) and mouse anti-β-tubulin (Sigma, St- Louis, MO).
The following secondary antibodies were used: Cy2-conjugated goat anti-mouse, Cy3-conjugated goat anti-rabbit, Cy3-conjugated goat anti-mouse, Cy5-conjugated goat anti-rabbit (all from Jackson Immuno Research, West Grove, PA); horseradish peroxidase(HRP)-coupled goat anti-mouse and HRP-coupled goat anti-rabbit (both from Dianova, Hamburg, Germany).

Mice were anaesthetized with 250 mg kg⁻¹ avertin and transcardially perfused with 0.9% saline, followed by 4% paraformaldehyde dissolved in 0.1 M phosphate buffer saline (PBS). Brains were harvested, fixed for 4 h in 4% paraformaldehyde, and immersed in 30% sucrose for 2 days. The brains were cut into 30-μm coronal sections on a cryostat (Leica CM1900, Germany). Floating sections were washed for 5 min 3 times in PBS with 0.3% Triton X-100 (PBST), and then blocked by 3% Bovine Serum Albumin in PBST. Subsequently, sections were incubated with primary antibody in PBST overnight at 4 °C. After washing in PBST, sections were incubated with secondary antibody in PBS for 1 h at room temperature. After washing again, sections were cover-slipped 50% glycerol mounting medium. Image data were acquired and digitalized using a confocal microscope (Zeiss LSM510 Meta, Germany). Primary antibodies used were: rabbit-anti-GFP (1:500, thermos fisher scientific), chicken-anti-GFP (1:500, Abcam), rabbit-anti-TH (1:500, Merk Millipore), and rabbit-anti-TPH2 (1:500, Merk Millipore). Secondary antibodies used were: fluorescein-conjugated goat-anti-rabbit (1:500, Jackson ImmunoReseach), fluorescein-conjugated donkey-anti-chicken (1:500, Jackson ImmunoResearch), and Cy3-conjugated goat-anti-rabbit (1:500, Jackson ImmunoResearch).

SC spreads were prepared via drying-down technique described by Peters et al.^{34 (link)}. Immunofluorescence staining was performed according to the protocol by Anderson et al.^{35 (link)}. Prior to immunostaining, slides were incubated in a solution of 10% PBT (PBS with 3% bovine serum albumin and 0.05% Tween 20) and 90% PBS for 45 min to reduce non-specific antibody binding. Primary antibodies included rabbit polyclonal anti-SYCP3 antibodies (1:500; Abcam, ab15093), human anticentromere antibodies (1:100; Antibodies Inc., 15-234) and mouse monoclonal anti-MLH1 antibodies (1:30, Abcam, ab14206). The slides were incubated with antibodies overnight in a humid box at 37 ℃, and then washed three times in PBS with 0.1% Tween 20 for 15 min each time. Secondary antibodies included Cy3-conjugated goat anti-rabbit (1:500; Jackson ImmunoResearch, 111-165-144), FITC-conjugated donkey anti-human (1:100; Jackson ImmunoResearch, 709-095-149) and FITC-conjugated goat anti-mouse (1:30; Jackson ImmunoResearch, 115-095-003). The slides were incubated with them for 1 h under the same conditions. After washing, the slides were mounted in Vectashield medium with DAPI (Vector Laboratories, cat No. H-1000-10) under the coverslips. Microscopic analysis and image processing were performed as described previously^{36 (link)}.

Drosophila brain fixation and antibody staining were carried out as described previously [51 (link)]. Briefly, adult brains were dissected in phosphate-buffered saline (PBS), then fixed with 4% formaldehyde and washed a few times with PBT (PBS + 0.1% Triton X-100). After blocking with 3% BSA (in PBT), Drosophila brain samples were incubated in primary antibody (rabbit anti-TH; Sigma-Aldrich; T8700, 1: 1000 dilution) overnight at 4 °C with rotation, washed with PBT and incubated in secondary antibody (Cy3-conjugated goat anti-rabbit; Jackson Immunoresearch; 1:500). Samples were washed a few times with PBT, incubated with Vectashield and TH-positive DA neurons were analyzed by Carl Zeiss Upright Confocal Microscope (Zeiss).