We routinely quench RBC auto-fluorescence with the following procedure: 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 5 times for 10 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 8 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. -----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of mesruh turkekul Sent: Thursday, December 24, 2009 10:20 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Re: Histonet Digest, Vol 73, Issue 33 Hi Naira, The RBCs have a heme groups that have very high autofluorescence. They will glow with every filter in every color. They will glow even without any staining. Just try to image unstained section and you will see. "*Autofluorescence* is the fluorescence<http://en.wikipedia.org/wiki/Fluorescence>of other substances than the fluorophore <http://en.wikipedia.org/wiki/Fluorophore> of interest. It increases the background signal.Autofluorescence can be problematic in fluorescence microscopy <http://en.wikipedia.org/wiki/Fluorescence_microscopy>. In most fluorescence microscopy, fluorescent stains (such as fluorescently-labeled antibodies <http://en.wikipedia.org/wiki/Antibody>) are applied to samples to stain specific structures. Autofluorescence interferes with detection of the resulting specific fluorescent signals, especially when the signals of interest are very dim - it causes structures other than those of interest to become visible. Depending upon the shape of the structures of interest and the other structures, it may not be obvious that this has occurred. In some microscopes (mainly confocal microscopes<http://en.wikipedia.org/wiki/Confocal_microscope>), it is possible to make use of different lifetime of the excited states of the added fluorescent markers and the endogenous molecules to exclude most of the autofluorescence." Unfortunately there is no way to avoid that. If you check online you may find people using: sodium borhydrate, 0.1M Glycine pH=3, sudan black or many other reagents to prevent autofluoresence but the success rate is very low. You may try to get rid of the red signal of the RBC with the imaging software. They have different spectra than Alexa594 and you can do spectral separation. Happy holidays! Mesru _______________________________________________ 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