I'm doing a large scale analysis of expression patterns in transgenic zebrafish and I am chasing after certain developmental stages. Therefore I need to fix the fish at that stage and once the microscope is available, I can image them. I do not want to destroy the signals and want to keep the tissue in a good condition.
Usually people fix it in 4%PFA (in PBS), but what follows afterwards, varies from paper to paper. Can someone suggest me a protocol that has worked for them?
To sum up: I need to fix and store transgenic zebrafish embryos (up to 5dpf) for image analysis withing 1 month post fixation.
THANK YOU!
You didn't say what signals you are trying to image. Gross morphology? Fluorescence? RNA probes?
Hi Elizabeth, I'm trying to detect GFP from the transgenic line. I have injected an oligo that is supposed to affect a gene expression and I'm trying to see if it has an effect on cilia biology (which are GFP labelled). Thank you!
For GFP imaging, short term (a couple of weeks) storage in 1% PFA in PBS at 4°C worked for me. I usually moved the embryos to ScaleU2 (http://www.nature.com/neuro/journal/v14/n11/full/nn.2928.html) during this period (it clears the embryos in a few days). GFP fluorescence is preserved super well in Scale even during long-term storage, as long as evaporation of the solution is prevented, but the downside is that the eyes of embryos tend to swell in this solution, so it's not useful if overall morphology is important.
Tissues develop some green autofluorescence if you fix them with PFA, but it bleaches really quickly compared to GFP, so it's not problematic.
If you have GFP already in the embryo, fix in 4% PFA-PBS overnight, then rinse in 1x PBS and store in the dark at 4°C. Do not store in fix, as this will increase background.
To prevent 'bugs' you can add some pen/strep to the storage solution (keeps for weeks) or 0.01% sodium azide (keeps for months/years). We have seen signals after 12-18 months and got some good images.
Note that after fixing you can also counterstain using whole-mount immuno-histochemistry for any other proteins, rhodamine-phalloidin (stains filamentous actin, which is abundant in cilia) and/or DAPI (stains nuclei). These look nice next to GFP, and if you are going to image anyway, why not add some more channels? :)
We transfer to 50%-70% glycerol-dI H2O or 100% CFM-Mount (which is compatible with glycerol). Put on your favorite slide/coverslip or glass-bottom Petri dish and scan.
Good luck with your experiment.
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