Hello,
I have fixed and obtained frozen sections of salamander embryos for fluorescence microscopy. The sections are 8um thick and the embryo used for the attached images was kidney bean shaped (sorry not super familiar with the development; helping someone else with this project), maybe stage #24 according to this chart: http://www.virginiaherpetologicalsociety.com/amphibians/amphibian-development/amphibian-development.htm
In the attached images, I only rehydrated the section and stained it with Hoechst. I then mounted using a glycerol-tris-n-propyl gallate mounting medium. Unfortunately, there is a lot of autofluorescence in both the TRITC and FITC channels and it's pretty bright. I didn't see nearly as much autofluorescence when I mounted in PBS alone, but I was hoping for a more permanent mounting medium. My experimental sample will require viewing the TRITC and DAPI channels so I really need to minimize this autofluorescence.
So my question is, what is the stuff autofluorescing and why does the glycerol make it worse? I would guess that it is yolk and did run across some info about it being autofluorescent but it would be nice to be certain.
Thank you!
Hi Blanca,
you're probably seeing autofluorescence from lipids. What body part is the one shown in the image? You might want to check protocols by Xenopus people. They have to deal with the yolk, which I guess is your problem.
Hope this helps!
J.
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