Donkey anti mouse cy5

Brains were dissected in PBS, fixed in 4%PFA for 30 min at room temperature, and cleaned of remaining tissue in 0.3% PBST. Following 3 × 10 min washes in PBST, brains were blocked in 5% normal donkey serum (NDS) and incubated with primary antibody in block at 4° overnight. Following 3 × 10 min washes in PBST, brains were incubated with secondary antibody in block for 2 hr at room temperature. Following 3 × 10 min washes in PBST, brains were mounted in vectashield. Primary antibodies included: Mouse anti-nc82 (1:1000, Developmental Studies Hybridoma Bank), Rabbit anti-GFP (1:1000, Molecular Probes). Secondary antibodies included: FITC donkey anti-rabbit (1:500, Jackson), Cy5 donkey anti-mouse (1:500, Jackson). Brains were visualized with a TCS SP5 confocal microscope and images processed in NIH ImageJ.

To assess surface expression of Spike in adenoviral transduced cells, 1.25 × 10⁶ Hs 729 T cells were plated in p100 and cultured overnight. After 24 h, cells were transduced with the different adenoviral constructs at MOI 100 for 48 h. Then, cells were washed with PBS and detached with StemPro Accutase (A11105-01, Life Technologies, Carlsbad, CA, USA) and stained with Fixable Viability Stain 510 (564406; 1:1000 dilution; BD Horizon, Franklin lakes, NJ, USA) for 5 min at 37 °C. Cell were centrifuged and resuspended in PBS-BSA (0,05%) and incubated with monoclonal antibodies against anti-S1(NTD) (E7M5X; 1:200 dilution; Cell Signaling Technology, Danvers, MA, USA) or RBD region (40592-MM117; 1:250 dilution or 40592-R0004; 1:100 dilution; Sino Biological, Beijing, R.P. China) for 30 min. Then, cells were centrifuged and washed with PBS-BSA (0.05%) twice and incubated with Cy-5 Donkey anti-mouse (715-175-150; 1:400 Jackson Immunoresearch, West Grove, PA, USA) or anti-rabbit (711-175-152; 1:400 dilution Jackson Immunoresearch) secondary antibodies for 30 min. Samples were then washed twice, resuspended in PBS-BSA (0.05%) and analyzed on a FACS Aria flow cytometer (Becton-Dickinson). The data were analyzed with FlowJo software (Becton-Dickinson) using gating strategy to evaluate only intact and viable cells (Supplementary Fig. 1a).

Optic fibres were implanted above the DRD in a separate cohort of TPH2-ChR2-YFP (n = 8) and WT (n = 3) animals to quantify c-Fos immunoreactivity. All animals underwent habituation to the toothless alligator clips and the recording chamber as previously described. However, these animals did not undergo MES. On the day following habituation, optogenetic stimulation (473 nm, 4 Hz, 10 mW, 5 ms) identical to the MES experiments was applied for 300 s. Animals were sacrificed 1.5 h after optogenetic stimulation to account for translation. Increased c-Fos in TPH2-ChR2 animals could support cell activation due to optogenetic light stimulation. Brain tissue was processed for IHC, as previously described. Primary antibodies: mouse anti-TpOH (1:30 000; MAB5278; EMD Millipore), chicken anti-GFP (1:3000; A10262; Invitrogen) and rabbit anti-cFos (1:1000; ABE457; EMD Millipore). Secondary antibodies: Cy5 donkey anti-mouse (1:500; 715175151; Jackson Immunoresearch), Alexa 488 donkey anti-chicken (1:500; 703545155; Jackson Immunoresearch) and Cy3 donkey anti-rabbit (1:500; 711165152; Jackson Immunoresearch). TPH2 expression was also quantified to ensure similar cell numbers existed in the TPH2-ChR2-YFP and WT sections used for quantification. One section was analysed per animal in a consistent 1.0 × 1.7 mm² area surrounding the DRN.

Adult fly brains were dissected in cold phosphate-buffered saline with 0.1% Triton-X (PBST) and fixed in 4% formaldehyde for 20–35 min. Brains were rinsed 3 X 15 min with PBST, blocked for 60 min in 5% normal donkey serum in PBST (NDST), and incubated for 24 hrs at RT in primary antibody diluted in NDST. Brains were then rinsed 3 X 15 min in PBST, incubated for 24 hrs in secondary antibody diluted in NDST, rinsed 3 X 15 min in PBST, cleared for 5 min in 50% glycerol in PBST, and mounted in Vectashield. Primary antibodies were as follows: rabbit anti-GFP 1:1000 (Molecular Probes A-11122), rat anti-RFP 1:1000 (Chromotek 5F8), mouse anti-HA 1:250 (BioLegend 901501), rabbit anti-SIFa 1:4000 (gift of J. Veenstra), and guinea pig anti-PERIOD 1:1000 (UPR 1140; gift of A. Sehgal). Secondary antibodies were as follows: FITC donkey anti-rabbit 1:1000 (Jackson 711-095-152), Cy3 donkey anti-rat 1:1000 (Jackson 712-16-150), Cy5 donkey anti-mouse 1:1000 (Jackson 715-175-151) and Cy3 donkey anti-guinea pig 1:1000 (Jackson 706-165-148). Immunolabeled brains were visualized with a Fluoview 1000 confocal microscope (Olympus). Trans-Tango flies were raised at 18°C and dissected ~2 weeks post-eclosion to maximize signal intensity [25 (link)].

Immunostaining was done on 60-μm free-floating coronal sections. Antibodies were applied in tris-buffered saline (TBS) with 3% donkey serum and 0.25% Triton X-100. Immunofluorescence was performed using anti GFP (rabbit polyclonal; 1:500; Invitrogen), anti NeuN (mouse monoclonal; 1:50; a gift from F.H. Gage, Salk Institute for Biological Studies, La Jolla, CA, United States), donkey anti-rabbit Cy3 and donkey anti-mouse Cy5 antibodies (1:250; Jackson Immuno Research Laboratories).