I have a question about some weird nuclei I see while imaging. I'm sorry if this isn't the best place to write it!
One image has bad, "shattered"-looking nuclei from dentate gyrus.
The other image has healthy nuclei from CA1.
I end up in some experiments with really terrible looking DAPI with "shattered" nuclei. Sections with this type of DAPI are highly correlated with really bad FISH staining as well. It seems like most of the tissue integrity is lost in general.
I've seen this before, but not enough times to know what step of tissue collection and processing could cause it. I don't *think* this is purely a cryosectioning issue.
Does anyone have any guesses for what could cause this issue? I'm guessing I can't be the first person to run into this!
Tissue processing
I am imaging coronal brain sections from mouse tissue that is flash frozen immediately after dissection. In this case this is imaging dentate gyrus, where nuclei should be exceptionally dense. 20 um cryosections are immediately placed onto Superfrost glass slides and stored at -70 (in this case for ~2 weeks).
Sections are fixed with 4% PFA on the slide and then dehydrated with serial EtOH incubations of 50%, 70%, and 100% for 5 minutes each. Then sections go through the RNAscope smFISH protocol, at the end of which they are stained with DAPI for 30 seconds.
Looks like an artifact from slow freezing of the tissue causing ice crystal formation in the cells and subsequent expansion and destruction. I've seen this many times, mainly from tissues obtained already frozen elsewhere. Hopefully, you are sure that the flash freezing is being done correctly.
Brett M Connolly I just re-processed some tissue from the same brains as shown above. The tissue quality looked excellent. The fixation, protease treatment, and dehydration steps were all the same.
The only difference was that the tissue that ended up looking poorly was put through the RNAscope protocol, whereas the tissue I just processed was mounted after dehydration.
So it seems like something is going wrong during the RNAscope protocol itself. I suspect it could be harsh washes with their detergent wash buffer, but I'll need to see.
It's definitely not a freezing issue, though, since these were from the same brains.
- Badges
- Science topic
- Similar topics
- Papers