Alexa 647 conjugated donkey anti rabbit igg

Antibody staining was executed as previously described^{20 (link)} using either zebrafish embryos or sections. The primary antibodies, sources and dilutions used were anti-Col2A1 (II-II6B3; DSHB; 1:100), anti-eGFP (TP401; Torrey Pines; 1:500), zns-5 (AB_10013796; ZIRC; 1:200) and anti-SM22 alpha/Transgelin (ab14106; Abcam; 1:250). Secondary antibodies were all from Invitrogen and used at 1:500: Alexa 488-conjugated donkey anti-rabbit immunoglobulin G (IgG; catalogue number A-21206), Alexa 546-conjugated donkey anti-mouse IgG (catalogue number A10036), Alexa 647-conjugated donkey anti-rabbit IgG (catalogue number A-31573) and Alexa 647-conjugated donkey anti-mouse IgG (catalogue number A-31571).

Rat hippocampal neurons were cultured on poly-L-lysine-coated coverslips. After treatment with NMDA for 4 h, the cells were fixed with 4% (w/v) paraformaldehyde/0.1 M sodium phosphate buffer (pH 7.1) for 1.5 h at 4°C, permeabilized with blocking buffer (0.1 M sodium phosphate buffer containing 0.4% (v/v) Triton X-100, 2% (v/v) donkey serum, and 1% (w/v) bovine serum albumin) for 30 min at room temperature, and then labeled with anti-drebrin A/E (C-term) and anti-NeuN antibodies at 4°C overnight. Alexa 488-conjugated donkey anti-mouse IgG (Invitrogen) and Alexa 647-conjugated donkey anti-rabbit IgG (Invitrogen) were used as secondary antibodies. F-actin was stained with Alexa 555-conjugated phalloidin (Invitrogen). For quantitative analyses of the immunofluorescence data, images of 100 randomly selected NeuN-positive neurons were collected using a BIOREVO BZ-9000 microscope (Keyence, Osaka, Japan) with a 100× oil immersion objective, and the signal intensities of the circular areas (40 μm in diameter) covering both the cell soma and the apical dendrites were measured using Image J analysis software.

Primary antibodies used were monoclonal anti-synaptobrevin 2 (104211 from Synaptic system; 1:5,000); monoclonal α-tubulin (T5168 from Sigma; 1:5,000); rabbit polyclonal anti-CgA (EL-35)^{23 (link)} (1:500 for IF, 1:1,000 for Western blotting); goat polyclonal anti-CgA (sc-23556 from Santa Cruz Biotechnology inc; 1: 200); rabbit polyclonal anti-Myo1b (HPA 013607 from Sigma prestige antibodies; 1:200 for IF, 1:250 for Western blotting); mouse monoclonal anti-GM130 (610822 from BDBiosciences; 1:1,000); rabbit polyclonal anti-furin (Ab3467 from Abcam; 1:200); sheep polyclonal anti-human TGN46 (AHP500 from AbD serotec; 1:500); rabbit polyclonal anti-p34-Arc/ARPC2 (07-227 from Millipore; 1:400); goat anti-type III collagen (1330-01 from Southern Biotech; 1: 200). For IF, secondary antibodies used were Alexa 488-conjugated donkey anti-rabbit IgG; Alexa 594-conjugated donkey anti-rabbit IgG; Alexa 647-conjugated donkey anti-rabbit IgG; Alexa 488-conjugated donkey anti-mouse IgG; Alexa 488-conjugated donkey anti-goat IgG; Alexa 594-conjugated donkey anti-goat IgG; Alexa 594-conjugated donkey anti-sheep IgG; Alexa 647-conjugated donkey anti-mouse IgG (Invitrogen; 1:500). For Western blotting, anti-rabbit, anti-mouse and anti-goat secondary antibodies conjugated to horseradish peroxydase (Santa Cruz biotechnologies; 1:2,000) were used.