Hi everyone! I’ve been letting my slides dry for about ten minutes before cover slipping them for IHC…Someone recently mentioned this is not a good idea. I’ve spoken to a few other people, and I’m getting mixed answers. Some prefer letting things dry overnight, others coverslip right away with the slide still very wet with PBS.
What do you prefer - letting dry completely, not letting dry at all?
Hi @ShanTan
Thanks for asking these questions here. These are the little but important things it hard find an answer to.
What mounting medium are you using? I use Prolong Gold. Many people us Vectashield and some like Mowiol.
@tberta taught me to add the mounting medium when the tissue is still moist but all the excess liquid is gone. I use a Kim wipe to get every drop of leftover PBS. But I like the tissue to still be moistened. So I don’t let it air dry completely. Some say full drying increases background.
Tell you what - why don’t you do a side by side comparison and report back to us? It probably isn’t a huge difference I bet. A lot of biology is folklore that becomes fact because once you find something that works you don’t want to mess with the other possibilities. Maybe this is time with a clear superior option, but one needs to test it out.
I’ll definitely test this out and let you guys know if I find any differences between dried vs wet slides. Currently using Vectashield because I prefer quick setting mounting media, as opposed to Prolong Gold.
I’ve been experimenting with this lately because I was tired of my free-floating sections moving around on the slides when I added coverslip medium (Vectashield) and applied the slip. I found that letting the slides mostly dry after mounting freefloating spinal cord sections helped them stay in place better when coverslipping. However, I keep a close eye on the slides so they don’t get bone dry - just until most of the PBS is gone. I would probably never leave them drying overnight after IHC - maybe this would in fact be okay but I’m not courageous enough to try (I have also heard that overdrying increases background.) I have not yet had an issue where the dryness of the sections interfered with the quality of IHC staining.
For thinner sections that are directly mounted onto the slide at the time of sectioning (therefore all IHC steps are processed on the slide), I will let these dry a few minutes before medium + coverslip, but I let them still be quite wet with PBS.
If you are using the hardset mounting medium from Vectashield I think extra PBS on the slide may interfere with its ability to harden, so if this is the case then you may want to wait until the PBS is mostly gone before coverslipping. However I use the softset Vectashield on somewhat wet slides and just seal the whole thing up with nail polish and it seems to work fine.