Hi all,
I have a question regarding something odd I'm seeing with DAPI on tissue. I'm trying to isolate the issue to prevent it in the future.
I have sagittal brain sections on slides. For some background on preparation, these were fixed in 10% neutral buffered formalin, then switched to 30% sucrose until they sunk. Unfortunately, these were then embedded in OCT instead of paraffin per supervisor request (we usually just do FFPE in my company's department), so they were cryosectioned and mounted directly onto slides. I stained for my two markers, used an autofluorescence quencher at the recommended time, and added one drop of Fluoromount-G with DAPI (that hardens) onto each piece of tissue. I slowly coverslipped to prevent bubbles and verified the mounting medium spread over each piece of tissue.
After I imaged, I noticed DAPI is strong around the outside of the tissue but gets weaker in the middle area. I was thinking it could be an issue with the imaging because we use an automatic scanner (Axioscan), but I've also noticed something like this happen on brain tissues (not spinal cord, liver, or heart tissues, though) when I image on our Olympus confocal's widefield setting.
Has anyone had this issue before or any suggestions on where to go from here? I ordered a new mounting media just in case it happens to be that, but I don't think it's the mounting media since it's been fine with other tissues.
Thank you!
Hi Andrew,
This is the protocol I used for staining OCT-embedded mouse brains.
1. Remove OTC
2. Wash with PB
3. Wash with PBS
4. Block with H2O2, 0.3% 30 mins
5. Wash with DW
6. Wash with PBST
7. Block with normal goat serum in PBST (1:100), 1h
7. Incubate with primary antibody in 2% normal goat serum with appropriate dilution at 4oC, overnight.
8. Wash with PBST
9. Incubate with secondary antibody 1:50 in 2% of normal goat serum, 1h, room temp.
10. Wash with PBST.
11. Mount the slides with VECTASHIELD® PLUS Antifade Mounting Medium with DAPI, Vector laboratories, Catalog no. H-2000-10
Hope this helps!
Hi Andrew,
could DNA degradation in the center be the cause? How large are your samples? Is the fixation done by immersion or by perfusion and how long do you fixate?
Best regards
Henrike
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