How to block autoflourescence in formalin fixed tissue???

Jim Hutchins hutchins at fiona.umsmed.edu
Wed Dec 6 23:03:05 EST 1995


Kevin Henne (khenne at fred.fhcrc.org) wrote:
: I would like to analyze formalin fixed paraffin embedded murine tissue 
: using a double labeling system,(FITC & Texas Red). I'm looking for 
: methods to block autoflourescence in tissue fixed and processed in this 
: manner.

Don't use formalin :-).  Seriously, commercial-grade formalin has impurities
and stabilizers which cause fluorescence background.  Take 500 ml glass-
distilled or MilliQ water and heat to 60-70 deg C.  Add 40 g paraformalde-
hyde slowly with constant stirring (do this in the fume hood or you will
regret it :-) ).  Add 10 N NaOH dropwise until the solution clears; it
should not take more than 5-10 drops.  Now you have 8% formaldhyde.

This 8% stock will keep for several days at 4 deg C.  Add one part of it
to one part of your favorite 2X buffer (0.2 M sodium cacodylate, pH 7.4 is
my fave; 0.2 M phos or 0.1 M Tris is OK too).  This 4% formaldehyde soln
is your working solution, and you should never have any autofluorescence
problems with it.

Good luck


--
Jim Hutchins
Assoc Prof Anatomy, Asst Prof Neurology (Research), Univ Mississippi Med Ctr
http://fiona.umsmed.edu/~hutchins/    ***    E-mail:   hutchins at umsmed.edu
``It became necessary to destroy the town in order to save it.''--American
infantry officer firing on Ben Tre, Vietnam, 2/8/68



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