Biotin conjugated secondary antibody

Dechorionated Drosophila embryos were fixed in paraformaldehyde-heptane solution for 30 min, followed by devitellinization using methanol. Freshly fixed embryos were then labeled with digoxigenin (DIG)-labeled antisense probes following the standard procedure described in Knirr et al., 1999 (link). After rehydration and post-fixation in 10% formaldehyde, embryos were pre-hybridized for 1 hr at 55 °C. mRNA probes were added to the embryos which were then incubated for 16 hr at 55 °C. DIG present on the synthesized mRNA probes was subsequently labeled with anti-DIG primary antibody (Enzo; ENZ-ABS302-0001; 1:1,000) and biotin-conjugated secondary antibody (Invitrogen; 1:500). To detect fluorescent signals, embryos were incubated with AB solution (PK-6100, Vectastain Elite ABC kit) followed by the tyramide signal amplification reaction (Cy3 fluorescent dye in amplification diluent; 1:50) (Perkin Elmer Life Sciences, NEL753001kt). To mark the SG and the apical membrane, the embryos were immunolabeled with antibodies for CrebA (SG nuclei) and Crb (apical membrane).
Primer sequences used for mRNA probe synthesis in this study are listed below.

Based on our previous experiment (Liu et al., 2016 (link)), the mice were perfused transcardially with 0.9% saline followed by 4% paraformaldehyde (PFA) after deep anesthesia with pentobarbital. The brain was collected and dehydrated completely in 30% sucrose solution with 4% paraformaldehyde at 4°C. Brains were sliced coronally (30 μm thick) and stored at -20°C in cryoprotectant solution. Immunostaining was performed according to our previous method. In short, the brain sections were incubated with a rabbit anti-doublecortin (DCX) (1:1000, Cell Signaling) primary antibody in 1% bovine serum albumin (BSA) (12 h, 4°C). For BrdU staining, the sections were pretreated with 2 N HCl for 30 min at 37°C and then incubated with mouse anti-BrdU (1:500, BD Biosciences). After that, the sections were incubated with biotin-conjugated secondary antibody (1:200, goat anti-rabbit; Invitrogen) or Cy3 (1:500; donkey anti-mouse; Jackson ImmunoResearch) (2 h, 37°C), then treated with avidin-biotin peroxidase complex (Dako) for immunohistochemistry or 4′, 6-diamidino-2-phenylindole (DAPI, Beyotime, China) for immunofluorescence. The BrdU- or DCX-labeled cells were observed and photographed with a Zeiss (Oberkochen, Germany) Axivert microscope equipped with a Zeiss AxioCam digital color camera connected to the Zeiss AxioVision 3.0 system.