[Histonet] RE: reduction/elimination of red cell autofluorescence on FFPE sections for IF examination

[James Watson]

The copper binds with the hemoglobin in the RBC and greatly reduces or eliminates autofluoresence depending on timing.

10 mM Copper Sulfate

10 mM Copper Sulfate
Cupric Sulfate...............1.25 gm
50 mM Amonium acetate (pH5)........500.0 ml
Adjust ph to 5.0 with 1.0 M NaOH

50 mM Ammonium acetate (pH5)
Ammonium acetate.............1.93 gm
Distilled water................500.0 ml
Adjust pH to 5.0 with 1.0 M HCl

1.	After staining wash slides in Reaction Buffer 2 times for 15 minutes each.
2.	Rinse in PBS 2 times for 10 minutes each.
3.	Rinse in distilled water 5 minutes.
4.	Place slides in 10mM copper sulfate for 5 minutes.
5.	Return slides to distilled water and check for autofluorescence with microscope.
6.	if needed return slides to 10mM copper sulfate for a couple of more minutes and check again.
7.	Rinse slides for 5 minutes in distilled water.
8.	Rinse in PBS 2 times for 10 minutes each.
9.	Coverslip slides with appropriate mounting media.

James Watson HT  ASCP
Facilities Manager of Histology
GNF  Genomics Institute of the Novartis Research Foundation
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 Amy Porter
Sent: Monday, August 24, 2009 8:17 AM
To: Thurby, Christina; histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] RE: reduction/elimination of red cell autofluorescence on FFPE sections for IF examination

We have been utilizing 0.25% Ammonium Hydroxide(28%) + 70% Ethanol v/v for 1 
hour followed by 10 minutes in 50% Ethanol rinsing in several changes of 
buffer afterward and it seems to be working well for a multitude of 
autofluorescent problems on FFPE sections for us.  We tried to Sodium 
Borohydride and it was a little diffiucult to work with....quite hazardous.

Amy S. Porter, HT (ASCP) QIHC
Investigative HistoPathology Laboratory - Supervisor
2201 Biomedical Physical Sciences Bldg.  Rm #2133
East Lansing, MI  48824-3320
Phone:  (517) 884-5026
Fax:  (517) 432-1368
Email:  portera <@t> msu.edu
Web:  www.humanpathology.msu.edu
----- Original Message ----- 
From: "Thurby, Christina" <christina.thurby <@t> bms.com>
To: "Thurby, Christina" <christina.thurby <@t> bms.com>; 
<histonet <@t> lists.utsouthwestern.edu>
Sent: Monday, August 24, 2009 11:00 AM
Subject: [Histonet] RE: reduction/elimination of red cell autofluorescence 
on FFPE sections for IF examination


Sorry I did not include my contact information.
Christina Thurby
christina.thurby <@t> bms.com
812-429-8097
Thanks!

>-----Original Message-----
>From: Thurby, Christina
>Sent: Monday, August 24, 2009 9:59 AM
>To: 'histonet <@t> lists.utsouthwestern.edu'
>Subject: reduction/elimination of red cell autofluorescence on FFPE 
>sections for IF
>examination
>
>Can anyone give feedback on reagents/procedures to use for 
>immunofluorescence to
>reduce/eliminate red cell autofluorescence on FFPE sections.  I am using an 
>indirect method
>of labeling for two antibodies (FITC and Texas Red).
>
>I have not tried 0.1% sodium borohydride.  Will this help for formalin 
>fixed specimens?  I
>have read that it is used for gluteraldehyde autofluorescence reduction. 
>If appropriate how
>long should this reagent be applied to the specimen and at what 
>temperature.

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