Re: GFP tissue preparation for confocal microscopy

Posted by Caroline Bass on
URL: http://confocal-microscopy-list.275.s1.nabble.com/GFP-tissue-preparation-for-confocal-microscopy-tp2680696p2684449.html

I’ve looked at fresh EGFP tissue “smears” under a fluorescent microscope, basically thin slices of fresh tissue coverslipped in buffer and smushed down. It was a really quick and dirty way to see what was happening before I processed a bunch of fixed tissues. It was incredible how much fluorescence is lost as the buffer dried out under the coverslip. I heard that dehydrating frozen sections before fixation was not good for GFP. By the time I got through all of my smushes, the first ones I looked at were losing a great deal of signal.

I know this isn’t a very scientifically valid way of evaluating dehydration, but it seemed like a good explanation.



On 4/23/09 12:15 PM, "Andrea Hooper" <anh2006@...> wrote:

Dear Caroline,

What do you mean by dehydration precisely? Do you dry your slides after sectioning?

Here is our ampoules info:
(1) http://www.alfa.com, Alfa Aesar, Item #43368
(2) http://www.emsdiasum.com, Electron Microscopy Sciences, Item # 15710

Thanks,
Andrea


At 12:05 PM -0400 4/23/09, Caroline Bass wrote:

I concur. I regularly perfuse rats and mice with formaldehyde, and I don't have any problem getting a signal (viral vector delivered EGFP in brain, spleen, liver, muscle, etc.). I have seen severe loss with any sort of dehydration or methanol fixation in fresh tissue.
Can someone recommend a good PFA? (Product number and company please!) The ampules sound interesting...