I am trying to image my zebrafish embryos from 0-120hpf and am struggling with suitable mounting without damaging the specimen. Does anyone have a good protocol or own experience for it?
I am using Leica TSC SP8 motorised inverted DMI 6000 microscope.
Thank you
Since you are using an inverted microscope, make sure there is a cover slip at the bottom - do not use the usual microscope slides. You can make a hole in a small plastic petri dish and attach a cover slip to its bottom surface. Multi-well plates or chamber slides work fine too, since they are designed to work with inverted microscopes. I had good results with low-melting point agarose, as suggested above. It is easy to change the surrounding medium with it without displacing the embryos (particularly useful if you are planning on using a clearing solution). However, if your embryos are alive, the resistance of the agarose can prevent normal development. Some people use methylcellulose, but one needs to be careful to prevent any evaporation of water from it. It dries out quickly.
Otherwise, here is a protocol:
zebrafishbrain.org/pdfs/Mounting%20Techniques.pdf
and there is also this, if it helps:
http://dev.biologists.org/content/139/17/3242.short
You could always try to mount the embryos in low-melting agarose gel made with teleost Ringer's.
You can try mounting in glycerol which should give you extra protection against damage.
Please see http://www.zfic.org/common%20techniques/glycerol.html for detailed protocols.
Regards
Dear Monika,
Another option may be to build a small well on your glass slides either using a Dako pen or simply nail polish to the height of your embryos. That way the walls of the well will be high enough to protect your tissues from being squeezed when placing coverslip over the the tissue. In this method you can use your usual mounting medium.
best wishes,
Refik
Dear Monika
I wish I can help you but it is not my field.
Regards
Saeed
Since you are using an inverted microscope, make sure there is a cover slip at the bottom - do not use the usual microscope slides. You can make a hole in a small plastic petri dish and attach a cover slip to its bottom surface. Multi-well plates or chamber slides work fine too, since they are designed to work with inverted microscopes. I had good results with low-melting point agarose, as suggested above. It is easy to change the surrounding medium with it without displacing the embryos (particularly useful if you are planning on using a clearing solution). However, if your embryos are alive, the resistance of the agarose can prevent normal development. Some people use methylcellulose, but one needs to be careful to prevent any evaporation of water from it. It dries out quickly.
Otherwise, here is a protocol:
zebrafishbrain.org/pdfs/Mounting%20Techniques.pdf
and there is also this, if it helps:
http://dev.biologists.org/content/139/17/3242.short
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