Living colors

IHC of embryonic zebrafish hearts for quantification of cardiomyocytes was performed as previously described^{38 (link)}. Primary antibodies used were: Amhc (Atrial myosin heavy chain; University of Iowa Developmental Studies Hybridoma Bank) and Living Colors® polyclonal anti-DsRed antibody (Clontech). Secondary antibodies used were anti-mouse IgG1-FITC (Southern Biotech) and anti-rabbit IgG-TRITC (Southern Biotech). A Zeiss M2BioV_12 Stereo microscope was used to image hearts. Additional antibody information is included in Supplementary Table 2.

Immunohistology on cryosectioned brain sections was performed as described (Rink and Wullimann, 2001 (link)). We used only antibodies with previously validated specificity including rabbit anti-calretinin (Swant, catalog# 7697/1:1000), mouse anti-parvalbumin (Millipore 1:5000), rabbit anti-γ-aminobutyric acid (GABA, Sigma, catalog# A2052, 1:5000), rabbit anti-substance P (SP; immunostar, catalog# 20064/lot#1003002, 1:2000), mouse anti-tyrosine hydroxylase (TH; 1:1000; catalog# 22941; lot# 1241002) Immunostar; catalog# 20066; lot# 1301001), 1:1000), chicken anti-GFP (1:1000; molecular probes/invitrogen, catalog# A10262; lot# 1729643), rabbit anti-GFAP (1:100, Immunostar), rabbit anti-DSRed antibody (Living Colors, Clontech, Cat# 632496, 1:1000). Secondary fluorescence-coupled antibodies (Invitrogen): goat anti-chicken Alexa Fluor 488, goat anti-rabbit Alexa Fluor 488/555, goat anti-mouse Alexa Fluor 488/555. We used a total of 65 adult (unsexed) zebrafish fish and stained with maximally three antibodies at the same time so that each pattern was represented by at least three samples.

For immunoblotting, cells were grown to mid-log phase and cells were collected and resuspended in 5 ml of buffer E (50 mM sodium citrate, 100 mM sodium phosphate, pH 6.0, and 0.8 M sorbitol). Protoplasts were generated by incubation with Glucanex (1 mg/ml; Sigma-Aldrich) and Zymolyase-100 T (3 mg/ml; ICN Biomedicals) for 1 h. Then, the protoplasts were resuspended in lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 1% Triton X-100, 1 mM DTT, 1 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin). Protein concentrations were determined using the BioRad Protein Assay kit (BioRad). 50 μg of protein extracts were resolved by SDS–polyacrylamide gel electrophoresis (PAGE) on 10% gels and probed with anti-GFP (JL-8 Living Colors, Clontech), polyclonal DsRed (Living Colors, Clontech), anti-HA Rat monoclonal antibody (3F10, Roche) or anti-tubulin (Sigma 75168). Protein transfer, blotting, and chemiluminescence detection were performed using standard procedures ^{49 (link)}.

Flag (mouse monoclonal, Sigma, Cat no. F1804); GFP (chicken IgY, Avés, Cat no. GFP-1020); dsRED (rabbit polyclonal, Living Colors, Clontech, Cat no. 632496); MAP2 (mouse monoclonal, Sigma, Cat no. M1406); GM-130 (rabbit monoclonal, Abcam, EP892Y, Cat no. ab52649); neuropilin-2 (Nrp2) (rabbit polyclonal, Cell Signaling, Cat no. 3366S); Nrp2 (goat polyclonal, Research & Development, Cat no. AF2215); PlexA3 (rabbit polyclonal, Abcam, Cat no. ab41564), neuropilin-1 (Nrp1) (rabbit, Abcam, Cat no. ab81321); Nrp1 (goat polyclonal, R&D, Cat no. AF566); PSD-95 (mouse, NeuroMab, Cat no. 75028); SAP102 (mouse monoclonal, NeuroMab, Cat no. 75-058); neurofilament (2H3) (mouse monoclonal, Developmental Studies Hybridoma Bank); Ctip2 (rat monoclonal, Abcam, [25B6], Cat no. ab18465).

Rabbit anti-green fluorescent protein (GFP; Torrey Pines Biolabs, Cat# TP401, dilution 1:1,000), rat anti-GFP (Nacalai Tesque; Cat# GF090R, dilution 1:1,000), mouse anti-bromodeoxyuridine (BrdU, Roche; Cat# B8434, dilution 1:300), mouse anti zona occludens 1 (ZO1, Invitrogen; Cat# 33-9100, dilution 1:300), mouse anti-acetylated tubulin antibody (IgG2b; α-tubulin; Sigma; Cat# T7451, dilution 1:250), rabbit anti-γ-aminobutyric acid (GABA; Sigma; Cat# A2052, dilution 1:1,000), mouse anti-synaptic vesicle protein 2 (IgG1; SV2; DSHB; Cat# AB 2315387, dilution 1:250), rabbit anti-DSRed antibody (Living Colors; Clontech; Cat# 632496, dilution 1:300), rabbit anti- red fluorescent protein (RFP, MBL; Cat#PM005; dilution 1:2,000).

Dissected gills were fixed in Bouin’s fixative and mounted in 1% low melting temperature agarose (Flowgen). For sectioning, gut was fixed in 4% paraformaldehyde, embedded in 25% fish gelatin/15% sucrose, and sectioned at 20 μm thickness on a Leica 3050 S cryostat. Immunohistochemistry was performed for enhanced GFP or mCherry according to standard protocols using rabbit polyclonal anti-GFP (1:500, Ab290; Abcam), mouse monoclonal anti-mCherry (1:500, Living Colors; Clontech), anti-rabbit Alexa Fluor 488 (1:500; Molecular Probes), and anti-mouse Alexa Fluor 594 (1:500; Molecular Probes). Whole-mount in situ hybridization was carried out as previously described (32 (link)). To generate the cd4-1 probe, an ∼1-kb fragment was cloned by RT-PCR using the following primers: forward, 5′-CGCGTCTCTCTATCAGCAGA-3′, reverse, 5′-CTGTTTGTGTCTGCGGATGT-3′.

Co-IP and BiFC experiments were performed using transient protein expression in N. benthamiana leaves infiltrated with Agrobacterium carrying corresponding constructs as described previously^{44 (link)}. For co-IP, a 15 µL volume of HA antibody (3F10, Roche) was added to 1 mL of total protein extract and incubated at 4 °C for 4 h with gentle shaking before 20 μL of pre-rinsed protein G agarose beads (Millipore) was added. The mixture was incubated for another 3 h at 4 °C. The beads were washed five times with PBS buffer, separated by SDS-PAGE, and subjected to immunoblotting using anti-GFP (Living Colors, Takara, Cat#632375, dilution 1:3000), anti-HA antibody (3F10, Roche, Cat#11867431001, dilution 1:5000), or anti-ubiquitin antibody (D9D5, Cell Signaling Technology, Cat#8081S, dilution 1:1000). For BiFC, leaves were collected for microscopic imaging 48 h after Agrobacterium infiltration. Confocal laser scanning microscopy was performed using a TCS SP8 STED confocal microscope (Leica). sYFP fluorescence was excited by the 514 nm Argon laser and detected using a custom 522 nm to 545 nm band-pass emission filter, whereas mCherry fluorescence was excited by the 561 nm laser and detected using a custom 595–620 nm band-pass emission filter.