Today we’re moving forward with the coral ISH probes we made yesterday. After the sea star probes didn’t work out yesterday (twice), we decided to save those for another time. Monica and Ruth are currently running PCR again with our two new primers to verify that there’s nothing wrong with the PCR in itself.
As for the ISH with the coral bacterial probes, we need to go through several steps to prepare histology slides for hybridization:
- Select slides!
- De-paraffinize slides with safeclear (a low-toxicity alternative to xylene)
- Rehydrate slide tissues with a descending series of ethanol: 100% EtOH (x2), 95%, 80% 70%, 50%
- Rinse in sterile H2O and equilibrate in Tris buffer
- Incubate in Proteinase K/Tris buffer (opens up tissues to allow our probe to bind to DNA)
- Incubate in prehybridization buffer (makes DNA more accepting of annealing to probes)
- HYBRIDIZE! WOOHOOOOOOOOOO
And then we’ll see tomorrow if they worked!