Novocrania fluorescent in situ for mesoderm

Started a Novocrania in situ with engrailed and pax258, two genes that are expressed in the mesoderm. I want to find out the exact location of these genes in the coelomic pouches. And why not a double in situ with both.

engrailed DNP pax258 DNP engrailed DNP + pax258 DIG

23/06/14

Added probes at 10:30.

25/06/2014

1st washing day. Everything normal until I was about to dilute the DNP antibody. There was 0.5 µL left and I needed 2 µL! So, passed embryos over to PTw and kept in the fridge until the new anti-dnp hrp arrives…

01/07/2014

Washing 5x with PBT and blocking for 1h. Adding the antibodies (anti-dnp 1:100 for engrailed and pax258 and anti-dig 1:250 for pax258 of the double) at 18h in 2mL eppendorf tubes.

02/07/14

Washed more than 5 times in PBT for around 10-15 minutes each. Then longer PTw washes, but not as long as 30 minutes. Started developing around 12h with TSA kit with Cy5 for the single fluorescent in situs and Cy3 for the double. Stopped the reaction with 3x 5min washes of detergent solution at 60 °C followed by 3x PTw and standard POD inactivation for the double.

Single in situs were washed a couple of extra times in PTw and stained with 1:5000 dilution of Sytox Green for 1h30. Embryos were washed in PTw and dehydrated in Methanol. Finally embedded in Murray’s Clear.

On the fluorescent scope the signal is not clear… there is a lot of background.

Incubated the double with the anti-dnp antibody overnight.