[Histonet] RE: Autofluorescence with FFPE tissue

[gayle callis]

You wrote:  We are trying to do do immunofluorescence on FFPE tissue
sections; we always get autofluorescence. We have tried Sodium Borohydride
treatment, does not work very well. Does anyone have good method to remove
this autofluorescence from FFPE tissue sections.

We have never tried Toluidine blue, Fe2/Fe3, Phosphomolbidic acid.

Thanks

Bader Siddiki, PhD

www.ImmunoBioScience.com

 

**********************************************

Bader, 

 

You may not be able to completely get rid of aldehyde induced
autofluorescence.  Another thing to try is 100 mM glycine in pH 7.4 buffer,
can be TRIS buffer or PBS.  After deparaffinizing and rehydrating into
water, place sections in this solution for 15 to 20 minutes, rinse and
proceed with staining. One can also soak the tissues prior to processing in
the same solution for an hour or so.  We have not tried the latter, but
working with sections has been successful with minimal, no more than 8 hour
fixation in NBF.  Minimal fixation may not be ideal since it may result in
incomplete fixation for your tissues. 

 

 

If autofluorescence persists, you can use a Near Infrared Fluorophore to
eliminate autofluorescence that way OR use the autofluorescence as a
"Counterstain" by using an extremely bright red fluorophore that resists
photobleaching e.g. Alexa 594 from Molecular Probes.  

 

Go to http://www.uhnres.utoronto.ca/facilities/wcif/fdownload2.html    You
can download a wonderful discussion on autofluorescence: causes and cures.
This document has more ways of eliminating autofluorescence or links for
doing so.   In this document, it tells how to do the sodium borohydride
treatment from a protocol written by Kramer along with Ian Clements
(Molecular Probes).  The solution works best when applied to the section
while the solutions it still "fizzing" and for PFA fixed tissue (very
similar to NBF fixed tissues), the solution is applied 3 times for 10
minutes each time to maintain freshness of the sodium borohydride.  There
was a recent discussion on Histonet on how to dispose of this chemical.  

 

OR you can use fresh tissue frozen sections fixed with cold acetone - as we
do.  We do not have autofluorescence problems when we do this.   

 

I have a synopsis of methods put on Histonet some time ago, and will be
happy to forward that to you as an attachment.  

 

Good luck

 

Gayle Callis

HTL(ASCP)HT,MT

Bozeman MT 59715