Re: Quenching autofluorescence

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From:"J. A. Kiernan" <>
To:Corazon Bucana <>
Content-Type:TEXT/PLAIN; charset=US-ASCII

On Tue, 8 Feb 2000, Corazon Bucana wrote:

> I am working with paraffin embedded samples, mainly archival specimens, and
> I am getting very intense autofluorescence of red cells and connective
> tissueswhen using the FITC or Rhodamine filter sets.  Has anyone tried
> quenching this autofluorescence?  I would appreciate any comments.

  Immersion of the slides in a dilute osmium tetroxide solution
  (0.5% is good; probably 0.1% or even weaker would be OK) for
  a few minutes, followed by a long wash (hours) in slowly running
  tap water suppresses autofluorescence very well. I can provide
  a few references (from 1950s to 1970s) if you are interested.
  This has been done mainly in connection with fluorescent techniques
  of carbohydrate histochemistry, including PAS variants and methods
  using fluorescently labelled lectins. I have done both myself. The
  most recent documented use of OsO4 to suppress autofluorescence
  that I have seen is in a Cambridge PhD thesis, 1986. Probably the
  work was later published but I haven't seen it - but then, I haven't
  looked. I do not know of anyone who has used OsO4 before doing
  immunofluorescent staining, but in principle it should be OK. 

  The suppresion of fluorescence is due to the presence of large atoms
  near the fluorescent molecules. You need an element that spreads itself
  evenly through the tissue, leaving a small residue after washing:
  enough to put down the weak autofluorescence but not enough to
  prevent the detection of the specifically bound fluorescent label.
  Mercury-containing fixatives have this effect, and were considered
  good for subsequent immunofluorescence in bygone years. From my
  experience I can say that paraffin sections of mercury-fixed brain
  have a dull green autofluorescence (blue excitation), whereas sections
  of formalin-fixed brain (which fluoresce quite brightly green when
  excited by a BG12 filter) have no autofluorescence after they have
  been dewaxed, hydrated exposed to osmium tetroxide and thoroughly 

  The presence of the nitro group (-NO2) opposes fluorescence. Objects
  fixed in Bouin or other picric acid-containing mixtures have much less
  autofluorescence than specimens fixed in aldehydes and quite a bit
  less than is seen after organic coagulant fixatives (Carnoy etc).
  Picric acid is trinitrophenol, so it delivers anti-fluorescence in
  spades. Perhaps enough remains in hydrated and washed paraffin sections
  to overcome the noise without drowning out the signal.

  Fluorescein-selective optics certainly don't completely remove the
  autofluorescent background. Filter/mirror blocks of the "rhodamine"
  type are much better. There isn't much stuff in paraffin sections of
  bits of higher animals that is excited by green and emits orange-red.
  If you must use a fluorochrome excited by near-UV or blue, chemical
  suppression of autofluorescence (picric fixative, OsO4 etc) could
  provide you with images that accurately portray the bound label
  that you want to study.

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1


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