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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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