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 jwat...@gnf.org -----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Amy Porter Sent: Monday, August 24, 2009 8:17 AM To: Thurby, Christina; histonet@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: port...@msu.edu Web: www.humanpathology.msu.edu ----- Original Message ----- From: "Thurby, Christina" <christina.thu...@bms.com> To: "Thurby, Christina" <christina.thu...@bms.com>; <histonet@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.thu...@bms.com 812-429-8097 Thanks! >-----Original Message----- >From: Thurby, Christina >Sent: Monday, August 24, 2009 9:59 AM >To: 'histonet@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. This message (including any attachments) may contain confidential, proprietary, privileged and/or private information. The information is intended to be for the use of the individual or entity designated above. If you are not the intended recipient of this message, please notify the sender immediately, and delete the message and any attachments. Any disclosure, reproduction, distribution or other use of this message or any attachments by an individual or entity other than the intended recipient is prohibited. _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet