Autofluorescence summary

Peter French p.french at ARNIE.CFI.UNSW.EDU.AU
Tue Apr 16 18:18:00 EST 1996

Thanks to all who contributed comments regarding autofluorescence
solutions. The replies are summarised below:

>I have found that using a counterstain works well.  In particular, after
>all the antibody incubations and washings we stain sections or cells with a
>1% Evans Blue solution in either PBS or distilled water for about 5', rinse
>briefly, and then mount.
>    The advantages are that the Evans blue seems to shift the
>yellowish-green autofluorescence to a wavelength that is then efficienctly
>blocked by barrier filters.  If you pull some of the barrier filters you get
>the apple-green fluorescein colour on a very bright red counterstain from
>the Evans Blue.
>    The drawback is that you can not double stain with any fluorochrome that
>emits in the red range (rhodamine or Texas Red) because the counterstain
>fluorescence is huge.
>    We have used this successfully to kill the autofluorescence in liver and
>in marine organisms.
>Warren Gallin (wgallin at

>I work with totally different material (plant tissues), but maybe the 
>information would be useful:
>for FITC: I stain the microtome sections (after the second antibody and 
>washing)with 0.01% Toluidine Blue in PBS (typically 10 minutes), which is 
>followed by 10 min washing in PBS and mounting in anti-fade mountant, 
>coverslips sealed by a nail varnish - i am quite happy with the folowing 
>  100 mg p-phenylendiamine in 10 ml phosphate-saline (0.01 M phosphate 
>buffer pH 7.4, 0.15 M NaCl)pH set to 8.0 with Tris pH 8.0. Add 90 ml 
>glycerol.  Store at -20 C, in darkness.
>  With this mountant, I get nice fluorescence of cytoskeleton (tubulin, 
>actin) even after > 3 weeks of storage at 4 C.
>With regards, 
>Stan Vitha (vitha.1 at 

3. Glutaraldehyde-induced autofluorescence can be treated thus:
You can try to incubate your cells with PBS containing 0.1% NaBH4 for 30 
>minutes. I find this technique in: Beisker W. et al. Cytometry 8:235-239(1987).
>Richard DELORME
>Laboratoire de Cytologie Analytique
>69373 LYON CEDEX 08
>E-mail : cytoana at

>If your specimens have been fixed with glutaraldehyde this invariably 
>gives rise to intense autofluorescence. You can get rid of it by 
>incubation of the fixed tissue section with 1% sodium borohydride in PBS 
>for 5-10min. It reduces the Schiff bases formed when the glutaraldehyde 
>reacts with amino groups in the tissue.
>Hope this helps
>Brian Burke (bburke at

Hope this is useful.

Peter French

Peter French, Centre for Immunology, St Vincent's Hospital, Sydney.
President, ANZSCBI

phone 61-2-361-2388
fax 61-2-361-2391
mobile 018-412-961

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