Hello all,
I have several slides of 10um-thick cryosectioned brain slices from a line of FosTRAP (tdTomato fluorescent) mice that I am attempting to counterstain with DAPI Fluoromount-G prior to fluorescence microscopy (Nikon Eclipse E800). The problem is, only the nuclei of the olfactory bulbs are showing the fluorescent blue color of the DAPI stain.
The animals were perfused with PBS and 4% PFA/4% acrylamide, and postfixed in a PFA/bis-acrylamide/acrylamide/VA-044 solution, followed by a wash in PBST. They were initially intended for the CLARITY brain-clearing procedure, and spent some time an electrophoretic clearing machine before I realized that it wasn't working and pulled them. Afterwards, I decided to cryoprotect and section the tissues in order to preserve the data. The cryoprotection and coverslipping protocol was as follows:
1. Immerse tissue (hemibrains) in a series of sucrose solutions (10%-20%-30%) until they sink
2. Place tissue in cryostat mold and cover completely with OCT, removing all air bubbles
3. Flash-freezing in a bowl of isopentane placed in liquid nitrogen
4. Storing at -80 C, wrapped in foil, until ready for cryosectioning
5. Acclimating to cryostat temperature (-20 C) for 1.5 hours before sectioning (10um)
6. Storing slides in -80 C until ready for coverslip protocol
7. Prior to coverslipping, removing slides from freezer and rinsing in PBS and water
8. Placing 4-5 drops of DAPI Fluoromount-G on the slide, lowering the coverslip, pulling off excess DAPI, and sealing with clear nail polish
I'm certain that the excitation/emission filters on the microscope are correct, and I'm confident in the protocols described above. I just can't seem to figure out why the DAPI stain isn't working for the hemibrain slices. It's been a problem for all of the slides, regardless of the animal they came from. I worry that the initial (failed) CLARITY protocol, which caused the tissue to swell somewhat, might have contributed to the formation of ice crystals during flash-freezing and tissue shattering, but there's little evidence for that when I check under the microscope -- and that would also mean that the olfactory bulbs would have been affected, which doesn't seem to be the case.
Does anyone have any thoughts? Thanks in advance.
Hi, Katherine.
The tissue section may have high sucrose inside of the nuclei. Probably wash several times with enough PBS+0.02%Tween 20 (PBT) for 10 min each to allow the remaining molecules diffuse away. Incubate the section in 20ug/ml DAP for 30 min followed by washing with enough PBT for 30 min to clear background. You should use enough DAPI beause they are not monolayer cell sheet.
Regards.
Some thoughts... DAPI usually doesn't label for one of the following reasons:
1) The tissue isn't permeabilized well (try adding a Triton perm step, followed by PBS wash, prior to mounting)
2) The issue isn't rehydrated well (leaving OCT there which can block dye entry (try a more thorough rehydration prior to mounting)
3) The DNA is degraded (I don't know if your Clarity protocol or fixation protocol may be doing something here)
4) There is something wrong with the mountant (you can test this by just mounting some fixed and permeabilized cultured cells)
5) The nucleic acids are bound by some other, competing molecule or dye (I'm doubtful on this one)
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