PLEASE HELP: IN SITUs - (Jun/07/2009 )
I am doing in situ hybridization with zebrafish embryos. The protocol calls for rinsing in MeOH and storing the fixed embryos in MeOH overnight at 4C. I forgot to do the "storing" part although I did the rinsing portion and went right ahead to hydrating and the procedure.
What do I do? Is it ok if I skipped the "storing" part? How will it affect my results? Why does one use MeOH?
It will be OK. The methanol is a fixative, it maintains the proteins and DNA/RNA intact and preserves structures so that you can detect with your probes and see where it is localized. Fixing initially with paraformaldehyde is best, as it is better at preserving the structure, however for storing, you need to transfer the embryos to methanol as the formaldehyde degrades over time. Serial dehydration in increasing methanol percentage is the way to go. Dunking them straight into 100% MeOH dehydrates them too fast, causing the morphology to change.