Alexa fluor 488 and 594 conjugated secondary antibodies

Brain slices were washed with PBS for 20 min at room temperature, followed by antigen retrieval with 10 mM sodium citrate buffer at 85 °C for 30 min. The slices were washed with PBS followed by permeabilization with 0.4% Triton X-100 in PBS at RT for 20 min. Then, the slices were blocked with 10% donkey serum and 0.1% Triton X-100 in PBS at RT for 3 h followed by incubation with primary antibodies, 5% donkey serum, and 0.1% Triton X-100 in PBS at 4 °C overnight. After washing with 0.1% Triton X-100 in PBS at RT for 15 min three times, secondary antibodies, 5% donkey serum, and 0.1% Triton X-100 in PBS at 4 °C were added for 2 h. The slices were counterstained with DAPI and mounted with mounting media (Vectashield, Vector Laboratories Inc., Burlingame, CA, USA). The following antibodies were used: rabbit anti-TREK-1 (Alomone Labs, Jerusalem, Israel, 1:200; RRID: APC-047, 1:200), rat anti-GFAP (ThermoFisher, Waltham, MA, USA; 1:500, RRID: 13-0300, 1:500), and Alexa Fluor 488- and 594-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA; 1:300). All images were acquired using a Nikon Ti2 confocal microscope (Nikon Instruments Inc., Melville, NY, USA).

Embryos were fixed overnight at 4°C in 4% paraformaldehyde (PFA) and were washed with PBT-2 (PBS containing 0.5% Triton X-100) three times followed by incubation in blocking solution for 1 h at room temperature. Primary antibodies were then added to this blocking solution and incubated overnight at 4°C with rocking. The following antibodies were used as primary antibodies: anti-GFP (1:1000 dilution; Abcam, Cambridge, UK), anti-cleaved caspase-3 (1:500 dilution; Cell Signaling Technology Inc., Danvers, MA, USA), and anti-E-cadherin (1:200 dilution; BD Biosciences,USA). After three washes with PBT-2, Alexa Fluor 488– and 594–conjugated secondary antibodies (Jackson Immuno Research, West Grove, PA, USA) were all used at 1:500 dilution and incubated overnight at 4°C with rocking. Nuclei were labeled with 4,6-diamidino-2-phenylindole (DAPI; 1:1000 dilution; Invitrogen, Carlsbad, CA, USA) for 20 min at room temperature. For image collection, Z-sections were taken at 1 μm intervals through the depth of the primordium/neuromast. Maximum-intensity projections were generated for analysis, and images were processed using Photoshop software (Adobe). Cell counts were performed at the time of imaging by viewing the images under a fluorescence microscope (Eclipse; Nikon Instruments) using a 40× objective.