Alexa 647 goat anti rabbit

Fish measuring ~2 cm in length were euthanized by overdose in tricaine (Sigma A5040), and then fixed at room temperature in PBS containing 4% paraformaldehyde. Following washing and permeabilization with PBS containing 0.1% Triton X-100, LC3B antibody (Abcam ab48934) was diluted 1:200 in PBS containing 0.1% Triton X-100 and 1% bovine serum albumin (Sigma A1470). For labeling β-catenin, the primary antibody (BD Bioscience 610153) was diluted 1:200, while cleaved Caspase-3 (Cell Signalling Technology 9661) and ∆Np63 (Santa Cruz SC-8341) were diluted 1:100. The secondary antibody, Alexa-568 goat anti-rabbit (Thermo Fisher A11011), or Alexa-647 goat anti-rabbit (Thermo Fisher A27040) was used at 1:1000. To label nuclei, fish were incubated in 1:5000 SYTO 9 (Thermo Fisher S35854) for 1 h. Three independent labeling experiments were carried out for each antibody.

At the end of the behavioral analysis, mice were perfused transcardially with ice-cold PBS with 5 U/mL sodium heparin (Hospira). Brains were post-fixed in 4% PFA for 48 h, cryo-protected in sucrose, and 8 µm coronal sections mounted on SuperFrost slides (Fisher Scientific; 12–550-15), were post-fixed in 4% PFA for 1 h, blocked in 2% bovine serum albumin, 10% normal goat serum and 0.1% saponin in PBS and incubated with rabbit anti-synaptophysin (1:1000, Millipore; AB9272) or rabbit anti-PSD95 (1:1000, Abcam; AB18258), followed by Alexa 488 goat anti-rabbit (1:500, Invitrogen; A-11043) or Alexa 647 goat anti rabbit (1:500, Thermo Fisher Scientific; A-21245). As a negative control, the primary antibody was omitted. Fluorescence was visualized using the Nikon A1R Confocal Microscope (Nikon Instruments Inc., Melville, NY, USA) using 40X objective. The average number of synaptophysin and PSD95 positive puncta were quantified in the CA1 of the hippocampus in three regions of interest using the spot detection feature of the Nikon NIS-Elements Advanced Research Software (Nikon Instruments Inc., Melville, NY, USA).

Rabbit anti-hERG1a (#12889 from Cell Signaling, 1:100), rabbit anti-hERG1b (#ALX-215–051 from Enzo, 1:100), rabbit anti-pan hERG (#ALX-215–049 from Enzo, 1:3000), rabbit anti Na_V1.5 (#ASC-005 from Alomone or #D9J7S from Cell signaling, 1:500), were used for immunofluorescence, western blot or RNA-IP experiments. Alexa 647 goat anti-rabbit, Alexa 488 goat anti-rabbit or Alexa 488 donkey anti-mouse were employed for indirect immunofluorescence or immunoblotting experiments (Thermofisher; 1:1000).

To positively identify focal adhesions in the endothelium, we injected 4% PFA into the blood stream of Tg(fli1ep:vinculinb-eGFP)^uq2al anaesthetised wild types at 50 hpf. Once cardiac contraction had stopped, the embryos were fixed whole mount in 4% PFA overnight and staining was performed as previously described (Lagendijk et al., 2011 (link)). Briefly, after fixing, embryos were washed in PBS-T (0.3% Triton-X100/PBS) three times, incubated in proteinase K for 10 min and washed again three times with PBS-T. Subsequently, embryos were incubated in blocking buffer (10% goat serum in PBS-T) for 1 h and with primary antibodies in blocking buffer at 4°C overnight. Washing and blocking steps were repeated the next day and embryos were incubated with secondary antibodies in blocking buffer at 4°C overnight. Secondary antibodies were removed the next day followed by three subsequent washes with PBS-T. Embryos were stored in PBS-T up to 1 week for imaging. Antibodies used were as follows: rabbit-anti-phospho-paxillin(Y118) (Thermo Fisher Scientific, 44-722G0, 1:200 dilution), chicken-anti-GFP (Abcam, ab13970, 1:200 dilution), Alexa 647 goat-anti-rabbit (Thermo Fisher Scientific, A32733, 1:500 dilution) and Alexa 488 goat anti-chicken (Thermo Fisher Scientific, A11039, 1:500 dilution).

Primary antibodies used for this study were Rab 11a (Invitrogen), Rab11b (Sigma) Rab 14 (Sigma), Rab 25 (Sigma), and RCP (Sigma). Additional details of primary antibodies including dilutions and Antibody Registration number are included in S1 Table. Secondary antibodies for immunofluorescence microscopy were Alexa 546 goat anti-chicken, Alexa 488 goat anti-mouse, and Alexa 647 goat anti-rabbit, all purchased from Life Technologies and used at a dilution of 1:400. For western blotting, IRDye800- and IRDye700- labelled goat anti-rabbit and goat anti-mouse secondary antibodies (Licor) were used at a dilution of 1:10,000.