Alexa fluor dye

Third-instar larval tissues were dissected and fixed with 3.7% formaldehyde in PBS for 20 min, followed by washes in PBS with 0.1% Triton X-100 (0.1% PBT). Primary antibodies used were anti-GFP (1:1000; A11122, Invitrogen) and anti-Tau (1:5000; 5A6, DSHB). Secondary antibodies conjugated with Alexa Fluor dyes (Thermo Fisher Scientific) were diluted 1:1000 in 0.1% PBT. Samples were mounted in 50% glycerol/PBS. Brains from 10-day-old flies were dissected in cold PBS and fixed in 3.7% formaldehyde in PBS for 30 min. Samples were washed three times in PBS with 0.5% Triton X-100 (0.5% PBT; Sigma-Aldrich) and incubated in blocking buffer (0.5% PBT+0.5% BSA+0.5% FBS) for 90 min at room temperature. Incubation with primary antibodies anti-cleaved Caspase-3 (Asp 175) (1:50; Cell Signaling Technology) and anti-ELAV (1:400; DSHB) was performed in blocking buffer overnight at 4°C. Secondary antibodies conjugated with Alexa Fluor dyes (Thermo Fisher Scientific) were diluted 1:1000 in 0.5% PBT and incubated for 3 h at room temperature. Tissues were mounted in Fluoromount-G™ Mounting Medium (Thermo Fisher Scientific). Samples were imaged on a Leica SP5 confocal microscope, and images were processed using ImageJ [National Institutes of Health (NIH)].

About 50 embryos collected within 3 h were put into an individual vial of fly food to avoid crowding and the larvae raised in an incubator at 25°C for appropriate lengths of time before dissection. Imaginal discs were fixed and stained according to standard protocols. The primary antibodies used were mouse anti-phospho-Histone3 (1:1000; Cell Signaling Technology), mouse anti-Mmp 1(1:30; a mixture of 5H7B11, 3A6B4 and 3B8D12, DSHB) and mouse anti-beta-galactosidase (1: 25, 40-1a, DHSB). The secondary antibodies conjugated with various Alexa Fluor dyes (Thermo Fisher Scientific) were used at 1:500. Phalloidin conjugated with Alexa Fluor dyes (1:1000, Thermo Fisher Scientific) and Hoechst 33342 (1:10,000, Thermo Fisher Scientific) were used to stain F-actin and DNA, respectively. All images were acquired on a Leica TCS SP8 confocal microscope.

Perinatal retrograde labeling of CPN, and CSMN was performed using a Vevo 770 ultrasound backscatter microscopy system (VisualSonics). Briefly, P1 pups of either sex were anesthetized by hypothermia, and CC, or pons, respectively, were injected under ultrasound guidance using a pulled glass micropipette (tip diameter, 80–100 μm), and cell bodies were labeled with the β-subunit of cholera toxin (CTB) labeled with Alexa Fluor dye (2 mg/ml, Invitrogen). For CSMN, six 23-nl injections of cholera toxin subunit B (2 µg/µl) were deposited bilaterally into the pons. The brains were harvested 2 d after injection (P3).
Because CStrPN reach the striatum later than P1, pups were anesthetized with hypothermia and injected sterotaxically at P3. The striatal injection point is very close to the anterior commissure in the developing brain, so both CStrPN and ACN were labeled simultaneously. The injections were made at an angle of 32.5° from horizontal, 3 mm posterior to bregma, and 1.8 mm lateral (left). The CTB labeled with Alexa Fluor dye (2 mg/ml, Invitrogen) was injected at a depth of 4.2 mm, and 4.6 nl of dye was delivered three times (total of 13.8 nl) into three injection sites at depths, 4.2, 4.1, and 4.0 mm. The needle was removed after 5 min to allow diffusion and avoid retracting the dye with the needle. The brains were harvested 2 d after injection (P5).