[Histonet] RE: IF on paraffin

James Watson JWatson <@t> gnf.org
Fri Sep 26 12:08:43 CDT 2014


We stain IF on paraffin all the time, typically about 100-200 slides per week .  We quench all of our fluorescence work with CuSO4.  Here is our CuSo4 quenching technique.  Since quenching depends on what is causing the autofluorescence we have other protocols using 2.0mM Glycine, Sodium Borohydride, and Sudan Black.  We use them rarely.

10 mM Copper Sulfate
	i)	This treatment is primarily for inhibition of autofluorescence in Red Blood cells, but helps decrease autofluorescence in 		connective tissue.
	ii)	10 mM Copper Sulfate
		(1)	Cupric Sulfate...............1.25 gm.
		(2)	50 mM Ammonium acetate (pH5)........500.0 ml
		(3)	Adjust pH to 5.0 with 1.0 M NaOH
	iii)	50 mM Ammonium acetate (pH5)
		(1)	Ammonium acetate.............1.93 gm.
		(2)	Distilled water................500.0 ml
		(3)	Adjust pH to 5.0 with 1.0 M HCl
	iv)	Treatment Procedure
		(1)	Rinse in PBS 2 times for 10 minutes each.
		(2)	Rinse in distilled water 5 minutes.
		(3)	Place slides in 10mM copper sulfate for 8 minutes.
		(4)	Return slides to distilled water and check for autofluorescence with microscope.
		(5)	If needed return slides to 10mM copper sulfate for a couple of more minutes and check again.
		(6)	Rinse slides for 5 minutes in distilled water.
		(7)	Rinse in PBS 2 times for 10 minutes each.
		(8)	Coverslip slides with appropriate mounting media.

James Watson HT  ASCP
GNF  Genomics Institute of the Novartis Research Foundation
Scientific Technical Leader II, Histology
Tel    858-332-4647
Fax   858-812-1915
jwatson <@t> gnf.org

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Houston, Ronald
Sent: Friday, September 26, 2014 9:41 AM
To: Histonet
Subject: [Histonet] IF on paraffin

Anyone doing this? Needs some pointers in balancing quenching autofluorescence and suppression of fluorescent signal


Ronnie Houston, MS HT(ASCP)QIHC
Anatomic Pathology Manager
ChildLab, a Division of Nationwide Children's Hospital www.childlab.com

700 Children's Drive
Columbus, OH 43205
(P) 614-722-5450
(F) 614-722-2899
ronald.houston <@t> nationwidechildrens.org<mailto:ronald.houston <@t> nationwidechildrens.org>

"One person with passion is better than forty people merely interested."
~ E.M. Forster

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