Dear fellow scholars,
We have a mouse line with eGFP and tdTomato labeled axons going from the brainstem to the spinal cord. We are going to clear the spinal cord (using iDisco) in embryos and some postnatal stages and image it, but I fear the fluorescent signal is to weak to see all the details of axons. Is it possible to enhance the signal using anti-GFP and anti-tdTomato antibodies in whole mount spinal cords? Are there some embryonic stages in where this is easy, but gets harder as the animal matures? Does anyone have any recommended protocols?
'Best regards,
Anders
I wouldn't see why not? I've used an anti-GFP to increase the signal with Alexa Fluor 488 in transgenic zebra fish. The biggest suggestion I would have is to a proper proteinase K incubation to allow the antibody to tag the tissue first.
Hi Anders,
Yes it is absolutely possible to enhance the signal using GFP and TdTomato antibodies. The authors of the iDISCO paper have posted a list of validated antibodies (including GFP and TdTomato ones) here: https://idisco.info/validated-antibodies/
Regarding a recommended protocol - I would suggest following something similar to the most up to date protocol here: https://idisco.info/idisco-protocol/
This protocol has suggested incubation times that get longer as the embryo matures. You will have to figure out what incubation times and dilutions of antibodies work best for your experiments but even the intact adult spinal cord clears pretty easily.
Best wishes,
Brett
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