We needed to make up some probes for the RLO-infecting phage, so it made sense to hold off on running ISH on our sea star probes until we could run the RLO batches with them. The control tube for the probe boiled off again in the thermocycler last night. The lid was closed but there was a tiny crack in the upper part of the tube. Since this is the second time it’s happened, we think that we’ve bought a low-quality batch of tubes that don’t hold up well to being pulled off the 8-tube line into smaller groups. After using some probe for the gel, there is under 1uL of control probe left. We’re hoping that this is enough, since we dilute it at a factor of 1:1000 to bathe the tissues on our slides.
To prep our slides, we washed off all the paraffin with SafeClear, a less toxic alternative to xylene, and then slowly rehydrated the tissues with different proportions of ethanol and water. While not taxing, it took a lot of waiting time. When the tissues were finally hydrated, we had to applied a prehybridization solution to ready the samples for binding with the probe DNA. To save on reagents, we needed to bathe only the parts of the slide containing tissue. We got to use a pap pen to create a hydrophobic boundary around the tissues. Watching the fluid not escape the ovals we’d drawn was very cool, even though it’s not a complicated phenomenon. Finally, we applied our diluted probes directly to the thin slices of tissue and left them to incubate overnight. Fingers crossed the probes bind and we get some visible staining that indicate where they’ve attached.