[Histonet] Re: Reduction of autofluorescence using glycine
gayle.callis <@t> bresnan.net
Fri Dec 12 16:08:45 CST 2008
To reduce aldehyde induced autofluorescence, you can use 100 - 300 mM glycine in pH 7.4 buffer. TRIS buffer or even Dulbeccos PBS will work. You rehydrate the section and then immerse into the glycine solution for 20 minutes, maybe even longer. Glycine works by getting rid (binding?) of free aldehyde groups. You can either treat the tissue prior to processing (after fixation) by immersing for an hour or so, but we simply did the glycine treatment on individual sections. It worked best for us when we did a short length fixation in NBF.
This has been discussed at length on Histonet in the past, so do an archive search. One person put a summary together on various methods and what worked best for him.
There are other methods for getting rid of autofluorescence although some are less successful than others and one is made from a chemical that is explosive. Try IHCworld website, fluorescence topics or Google access this discussion written by Wright Cell Imaging Faculty, Toronto Western Research Institute, titled: Autofluorescence, Causes and Cures, a must read on the subject.
Another trick is to use fluorophores in the near infrared range, the camera sees the fluorescence but no autofluorescence and you cannot see this red fluorophore with the naked eye. Alexa 750 will work if you have the filters and excitation wavelength available.
Gayle M. Callis
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