Alexa fluor 488 donkey anti mouse igg

After EFs stimulation, the EBs or the dissociated cells were fixed with 4 % paraformaldehyde and permeabilized with 0.2 % Triton X-100. Cells were exposed to blocking solution (10 % horse serum, 1 % bovine serum albumin (BSA)) for 20 min. All antibodies were diluted in PBS with 1 % BSA. The EBs were incubated with monoclonal primary antibody anti-nestin (Millipore, Billerica, MA), or polyclonal primary antibody anti-ChAT (Millipore, Billerica, MA) for 2 h at room temperature. The EBs were also labelled with Rhodamine Phalloidin (Life Technologies, Grand Island, NY). The dissociated cells were incubated with monoclonal primary antibody anti-neurofilament (NF) 160 (Sigma-Aldrich, St. Louis, MO) and polyclonal primary antibody anti-ChAT for 2 h at room temperature. The secondary antibodies were Alexa Fluor^® 594 Donkey anti-goat IgG and Alexa Fluor^® 488 Donkey Anti-Mouse IgG (Jackson ImmunoResearch, West Grove, PA). Nuclei were stained with DAPI.

The immunostaining protocol was performed as previously described [60 (link)]. Zebrafish larvae (6-dpf) were anesthetized and fixed overnight in 4% PFA and stored in 100% methanol at −20°C for at least overnight. The larvae were treated with cold acetone (−20°C) for 8 minutes, transferred back to 100% methanol, and then gradually rehydrated to PBS + 0.5% Triton x100 (PBTr). The samples were then digested with 10μg/ml proteinase K in PBTr for 40 minutes and fixed in 4% PFA for 20 minutes. Samples were then washed 3 times with PBTr and incubated for 1 hour in blocking solution (PBTr, 10% calf serum and 1% DMSO). The samples were incubated overnight at 4°C with primary antibody in blocking solution. The primary antibodies used were rabbit anti-zfAgRP1 (1:500), rabbit anti-zfAgRP2 (1:1000), and mouse anti-GFP (1:100, MBL International Cat# M048-3). The next day, the samples were washed 4 times, for 30 minutes each wash, in PBTr and then incubated for 2 hours with the secondary antibodies; Alexa Fluor 488 donkey anti-mouse IgG (1:100, Jackson ImmunoResearch) and Alexa Fluor 594 donkey anti-rabbit IgG (1:100, Jackson ImmunoResearch). The samples were then washed 6 times in PBTr and transferred to PBS for the following imaging steps.