I'd like to try the CLARITY method on some of my knock-in mice with fluorescent reporter molecules attached to their post synaptic proteins. It is impractical for me to set up the entire method when I don't know if it will kill the fluorescence, so it would be excellent if I could come and borrow someone's set up for a short period to test the method on my samples.
Although I am not in the UK, I have recently helped to set up the CLARITY method in our lab group and have successfully used it to clear and visualize CNS tissue in GFP mutant mice. We kept our samples shielded from light throughout the protocol and did not notice any decrease in the fluorescence. If you want to try the CLARITY protocol without committing to manufacturing the whole setup, I recommend trying passive clearing instead of ETC. We have done this with great success in our lab with our tissue samples. Instead of the ETC setup, after polymerization we just keep the samples in a small amount of clearing solution in a 37C water bath and simply change the clearing solution daily. With this technique we were able to achieve completely successful clearing of our tissue in 1-2 weeks (depending on the sample size). We leave the protocol unchanged otherwise. Let me know if you have any further questions!
under http://clarityresourcecenter.org you should find all the information needed. But Michelle is right with the ETC chamber. You need a workshop in your facility to build it. Or as you can find at http://clarityresourcecenter.org the Deisseroth Lab is now also offering a short hands-on workshop where you also build your own chamber - try to get a slot ;-)
Nevertheless, why trying only this procedure? Did you test some of the other clearing methods already?
I'm so sorry I didn't respond earlier - for some reason this thing hid your messages!
I have tried some other clearing methods, including SeeDB and a urea based method whose name escapes me right now. I found SeeDB useful but we were concerned about the processing temperatures so we wanted to give Clarity a go.
I tried the passive clearing and it did work, thank you for that tip Michelle!
Grazing emission X-ray fluorescence (GEXRF) is a new development in X-ray fluorescence analysis related to total-reflection XRF. An optical flat carrying the sample is irradiated at an angle of approximately 90° with an uncollimated polychromatic X-ray beam. The emitted fluorescent radiation of the sample elements is measured at very small angles u...