Whole Mount RNA in-situ Hybridization

The RNA in situ hybridization is a four day protocol and involves the following steps.

Day 1:

The chemically treated embryos were fixed at 24hpf in 4% paraformaldehyde and

kept at room temperature for 1-2 hours. They were then washed with PBST(0.1%

Tween 20 in PBS) and 50% methanol made in PBST and stored overnight at -20°c in

100% methanol.

Day 2:

The embryos were rehydrated to PBST(0.1% Tween20 in PBS) through 50% Methanol/

PTw. Then the embryos were rinsed with 1:1 PBST:pre-warmed hybridization wash

which was later replaced with pre-warmed hybridization mix for 1hour at 65°c.

Finally the embryos were treated with a mixture of pre-warmed hybridization mix

and DIG-labelled RNA probe overnight at 65°c.

Day 3:

After removal of the probe, the embryos were rinsed with pre-warmed hybridization

wash at 65°c a few times. This was later replaced with TBST at room temperature.

The mixture of TBST and 10% heat inactivated sheep serum was applied for 1 hour

to block the non-specific sites. After this, the embryos were incubated in a solution

containing TBST + 10% serum + 1:2000 dilution of AP-anti-DIG antibody for 4hours.

This was washed with TBST overnight at 4°c.

Day 4:

The chromogenic reaction was initiated by replacing the TBST with NBT/BCIP

(nitro-blue tetrazolium and 5-bromo-4-chloro-3’-indoylphosphate p-toluidine salt

which are substrates for alkaline phosphatise) solution with 0.1% Tween 20, the

incubation was done in dark and monitored carefully. When the color developed the

reaction was stopped by immediately.

The embryos were observed under the dissection microscope (Zeiss)and photographed with the microscope using camera (AxioCam, ICc Zeiss).