[Histonet] eliminating tissue autofluorescence


Hi all. I am a newcomer to histonet and first would like to express my thanks for the many helpful responses I received to a question I posted earlier.

Here's another question - we are doing a lot of immunofluorescence on formalin-fixed human tissue sections lately. We get quite a bit of background autofluorescence in several of the channels, on multiple tissue types, and we'd obviously like to avoid this. I came across a paper by a German group (sorry, don't have the reference handy at the moment) in which they claimed that by pre-bleaching the unstained slides using a medium-wattage light source (basically an aquarium grow-light)prior to staining they could eliminate much of this background fluorescence in the tissues. This seemed to make sense, so I tried several times with several different lamps but saw no real improvement, even with very long exposure times (days!). I even went so far as to remove our high-intensity light source from the fluorescence microscope and used this by exposing slides directly to the mercury lamp for times up to 30 minutes. Still no diminution of background signals. Perhaps re-hydrating the t
issue first will help, as photobleaching is, I believe, supposed to be mediated by the generation of free radical species and I'll try this next. Still, I am suprised that a close-up 30 minute exposure to the unfiltered mercury arc lamp didn't do anything...

Anyone else have any thoughts??

-Alan Meeker

Alan Meeker, PhD
Department of Pathology
Division of Genitourinary Pathology
Bunting-Blaustein Cancer Research Building  Room 153
1650 Orleans Street
Baltimore, MD 21231-1000
e-mail: ameeker@mail.jhmi.edu

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