I am trying to do both brightfield and flourescent photos of the heart, videos of the heart, and z-stack imaging for zebrafish embryos at 72 hpf. I use tricaine as an anesthetic and have been trying to position in LMP agarose. However, I can't seem to get the fish in the same position in order to obtain comparable data. Thanks for your help.
I've been a recent conversation on this topic over at: https://www.researchgate.net/post/How_best_to_immobilise_zebrafish_larvae_for_imaging
That conversation is mostly about long term imaging but may have some techniques for you to try. You are doing short duration anesthesia and imaging? What orientation are you trying to get? What percentage are you making your agarose? Have you tried methyl cellulose?
Thanks for the reference. Yes, I am doing short term imaging. I am trying to get a lateral view of the fish with the left eye up to view the bulb more clearly using an inverted confocal. Currently, I am using 1.2% agarose, but I will try 3% methylcellulose this week.
I have good luck with the methylcellulose. A higher percentage keeps them very still, allowing me to line up the eyes in a lateral orientation (I mostly work in brighfield). A gel-loading pipet tip (ones with the flat/scooped ends) or thin fishing line works well as a tool to orient the fish gently and quickly without damage.
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