Hi! I have a problem with the proper positioning of 4 dpf zebrafish (AB strain) for dorsal imaging under a stereoscopic microscope. After the treatment, some of them have yolk edema so it is not possible to place them perfectly straight, even in methylcellulose or other immobilizing media. Of course, they are already anesthetized with tricaine.
In this experiment, I plan to take dozen or more photos (length and angle measurements) therefore I need some wise solution to deal with it efficiently and quickly. Any tips?
I will appreciate any help!
Hi,
Maybe you can try mount your fish in 1.2% low melting agarose? You can adjust your samples to get a proper position, and once the agarose solidifies, fish cannot move, and you can do your imaging with them. Hope this can be helpful!
Low melting agarose or gelatin would be the sturdiest immobilizing media. You could also try positioning the fry on an injection tray - they should fit into the grooves pretty snugly (may get squished a little from the sides).
Hi Lukasz,
I use a homemade mold, beacuse I had similar issues with zfish orientation.
I made a homemade mold using 3 cover glasses (20mm x 20mm, glued each other) + 15-20mm pieces of glass capillary (1mm, normally used for microinjection, broken by hands) glued to the cover glasses.
Then, I usually use a small petri dish (35x10mm) in which I put 1.5ml of 1% agarose and, gently, I put up my homemade mold.
Once agarose is solid, you can leave the mold and use it to put your zfish into the groove done by the glass capillary.
Rather than tricaine, I use methylcellulose and I can turn my zfish without problems (dorsal or ventral), by using tiny tips.
I hope this could help you.
Good Luck
Pierluigi
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