Cyanine 3 cy3

Third-instar larvae were dissected in 0 Ca²⁺ HL-3 and immunostained^{71 (link)}. All genotypes were immunostained in the same tube with identical reagents, and then mounted in the same session. The following antibodies were used: mouse anti-Bruchpilot (BRP; nc82; 1:100; Developmental Studies Hybridoma Bank; DSHB); mouse anti-GluRIIA (1:100; 8B4D2; DSHB); guinea pig anti-GluRIID^{72 (link)} (1:1000); rabbit anti-DLG^{17 (link)} (1:5000); mouse anti-DLG (1:100; 4F3; DSHB); mouse anti-FK1 (1:100; Millipore 04–262); mouse anti-FK2 (1:500; BML-PW8810; Enzo Life Sciences); mouse anti-FLAG (1:500, F1804; Sigma-Aldrich); mouse anti-GFP (1:1000, 3e6; Invitrogen, Carlsbad, CA); mouse anti-pCaMKII (1:100; MA1–047; Invitrogen). Donkey anti-mouse, anti-guinea pig, and anti-rabbit Alexa Fluor 488- (715-545-150, 706-545-148, 711-545-152; Jackson Immunoresearch), DyLight 405- (715-475-150, 706-475-148, 711-475-152; Jackson Immunoresearch), and Cyanine 3 (Cy3)- (715-165-150, 706-165-148, 711-165-152; Jackson Immunoresearch) conjugated secondary antibodies were used at 1:400. Alexa Fluor 647 conjugated goat anti-HRP (123-605-021; Jackson ImmunoResearch) was used at 1:200.

OC explants or cells were fixed with 4% paraformaldehyde (Biosesang, Gyeonggi-do, Korea) for 15 min at room temperature and permeabilized using 0.2% Triton-X 100 in PBS (PBST). After blocking with 1% bovine serum albumin in 1× PBST, samples were incubated with primary antibodies at 4 °C for 24 h. The primary antibodies were anti-phospho-CREB (#9198, Cell Signaling), anti-total-CREB (#9197, Cell Signaling), anti-connexin 43 (#3512, Cell Signaling), and anti-SOX2 (1:500, Cell Signaling; #4900). Samples were washed thoroughly and incubated with secondary antibodies tagged with fluorescein isothiocyanate (FITC) or cyanine 3 (Cy3; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at room temperature. F-actin was stained using phalloidin–Texas red (Invitrogen, Molecular Probes, Carlsbad, CA, USA), and nuclei were counterstained with 4′6,-diamidino-2-phenylindole (DAPI; Invitrogen). Coverslips were mounted onto slides with mounting medium (Vector Laboratories, Burlingame, CA, USA). The immunostained cells were observed using a Zeiss LSM 700 confocal microscope (Carl Zeiss Meditec, Jena, Germany).