Donkey anti mouse alexa 488

Immunohistochemistry was performed as previously described20 (link). Antibodies used were: mouse monoclonal anti-GFP 1:250 (Millipore), rabbit anti-synapsin 1:1000 (Synaptic Systems)22 (link), rabbit anti-PSD95 (Abcam)23 (link), Cy-3 anti-rabbit 1:400, Alexa 488 donkey anti-mouse 1:400 (Invitrogen), and rabbit anti-goat Alexa 555 (Invitrogen). Double immunohistochemistry for GFP with anti-synapsin or anti-PSD95 was performed at 3 dpf as follows: embryos were fixed in 4% paraformaldehyde (PFA) in PBS for 1.5 hours at room temperature (RT) and washed briefly in PBS (3 × 5 min) with 0.1% Triton X-100 (PBST). Embryos were then blocked (PBS with 1% BSA, 1% DMSO, 2% goat serum, and 0.1% Triton X-100) for 3 hours at RT, and then incubated in blocking buffer with primary antibodies overnight at 4 °C. Embryos were washed with PBST, and incubated with secondary antibodies overnight at 4 °C.
For sectioning, stained larvae was cryoprotected in 30% of sucrose with PBS for at least 2 hr, rinsed briefly in Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, CA), and frozen in a Tissue-Tek O.C. ethanol/dry ice bath. Sections were 20 μm and mounted on the Superfrost Plus Microscope slides.

During the proliferation stage, fixed cells were stained for markers of stemness (sex determining region Y-box 2 (Sox2), Alexa 488 donkey anti-rabbit; 1:1000, Invitrogen; Nestin, Alexa 555 donkey anti-mouse, 1:1000, Invitrogen), proliferation (Ki67, Alexa 488 donkey anti-rabbit; 1:1000, Invitrogen) and apoptosis (CC3, Alexa 555 donkey anti-mouse, 1:1000, Invitrogen). During the differentiation stage, cells were stained for markers of neuroblasts and mature neurons (respectively, doublecortin (DCX), Alexa 555 donkey anti-rabbit; 1:1000, Invitrogen; microtubule-associated protein 2 (Map2), Alexa 488 donkey anti-mouse, 1:1000, Invitrogen), astrocytes (S100 calcium-binding protein β (S100β), Alexa 488 donkey anti-rabbit; 1:1000, Invitrogen), and again apoptosis (CC3; Alexa 555 donkey anti-rabbit, 1:1000, Invitrogen). All cells were labelled using DAPI dye, as in previous publications [22 (link), 25 (link), 28 (link), 37 –39 (link)]. The percentage of Sox2, Nestin, Ki67, DCX, Map2, S100β and CC3 positive cells over total DAPI positive cells was counted using an insight automated imaging platform (CellInsight) (Supplementary Fig. 1a–h for representative images). At least six independent experiments were conducted on independent biological cultures, and each sample was tested in quadruplicate.