Anti fluorescein pod antibody

Double fluorescent in situ hybridisations were performed with a combination of DIG-labelled (neurod and crx) and fluorescein-labelled (gnat1 and gnat2) riboprobes. The protocol was similar to that described in the previous section for cryosections, with the following modifications: incubation at 4°C overnight with anti-fluorescein-POD antibody (Roche, 11426346910; 1:2000) was followed by incubation for 1 h at room temperature in TSA Plus Fluorescein Solution (PerkinElmer); inactivation of the first peroxidase was for 30 min in methanol/3% H₂O₂; incubation at 4°C overnight with anti-DIG-POD antibody (Roche, 11093274910; 1:2000) was followed by incubation for 1 h at room temperature in TSA Plus Cyanine 5 Solution (PerkinElmer). Sections were imaged on a Leica TCS SPE confocal microscope.

In situ hybridizations on retinal cryosections and dissociated cells were performed exactly as described in Trimarchi et al. 2007. For the dissociated ISH, one probe was synthesized with a digoxigenin (DIG)-label and the other probe was labeled with fluorescein. DIG-labeled probes were detected using an anti-DIG-POD antibody (Roche, 1∶1000) and a Cy3 tyramide solution (PerkinElmer), while fluorescein-labeled probes were detected using an anti-fluorescein-POD antibody (Roche, 1∶1000) and an Alexa-488 tyramide (Life Technologies). Tyramide amplification (Life Technologies) was performed for 10 minutes for the DIG-labeled probe, followed by inactivation in 0.3% hydrogen peroxide and tyramide amplification for the fluorescein-labeled probe. The slides were fixed in 4% paraformaldehyde and mounted. Six independent fields were photographed and quantified for each.

Embryos were fixed at 72 hpf in 4% PFA at 4 °C overnight. Fixed embryos were dehydrated and rehydrated through a methanol/PBST series, followed by permeabilization with 10 µg/mL proteinase K for 30 min and refixation in 4% PFA for 20 min at RT. Apoptotic cells were labeled by TdT-mediated dUTP nick-end labeling (TUNEL) assay using in situ cell death detection kit (Roche), according to the manufacturer’s instructions. After washing with PBST, embryos were blocked with 5% serum in PBST for 2 h at RT, and incubated with anti-fluorescein-POD antibody (1:1000, Roche) and anti-MEF2 antibody (1:150, Santa Cruz Biotechnology) overnight at 4 °C. After several PBST washes, apoptosis signal was enhanced by TSA Plus Cyanine 5 and Flourescein System (Perkin Elmer), then embryos were incubated with goat anti-rabbit Alexa Fluor 568 (1:200, Invitrogen) for 3 h at RT. Later, embryonic hearts were dissected using two 22-gauge needles and transferred to slides, where the tissues were mounted in ProLong Gold Antifade Mountant with DAPI (Invitrogen). Both DNase I-treated positive control and negative control, without TdT enzyme, were included.