Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Kevin, I use Sudan Black (from Humason's Animal Tissue Techniques). It is not fluorescent itself and works wonders on quenching endogenous fluorescence, including from blood. The only issue is that the staining is done in 70% alcohol to keep the dye soluble. So depending on your other staining conditions, it might not work for you. Good luck. Helen McBride Helen J. McBride, Ph.D. Senior Scientist Inflammation Group Amgen One Amgen Center Drive MS:29-5-1 Thousand Oaks, CA 91320-1799 Phone: 805-313-5476 Fax: 805-447-1982 Email:[log in to unmask] _____ From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Rosemary White Sent: Thursday, July 12, 2007 2:19 PM To: [log in to unmask] Subject: Re: quenching of Alexa633 Hi Kevin, You could try toluidine blue or crystal violet, though I've only used these to quench stains fluorescing in the yellow-green. Or possibly basic fuschin (from Schiff's), which will fluoresce in the green-yellow but I think is not fluorescent with red excitation. No experience with any of them on animal cells.... cheers, Rosemary Dr Rosemary White [log in to unmask] CSIRO Plant Industry ph. 02-6246 5475 GPO Box 1600 fax. 02-6246 5334 Canberra, ACT 2601 Australia _____ From: Kevin Braeckmans <[log in to unmask]> Reply-To: Confocal Microscopy List <[log in to unmask]> Date: Thu, 12 Jul 2007 16:04:45 +0200 To: [log in to unmask] Subject: quenching of Alexa633 Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal <http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal> Dear list, We are using transferrin tagged with Alexa633 to label the clathrin-mediated endocytosis pathway of living cells. We wanted to use trypan blue to quench the fluorescence of the remaining Alexa-Tf on the cell membrane. However, this does not seem to work, contrary to e.g. bodipy or fluorescein. Do others also have found difficulties in quenching Alexa-dyes by tripan blue? Does someone know an alternative quencher which might work? Thanks for any suggestions, Kevin Kevin Braeckmans, Ph.D. Lab. General Biochemistry and Physical Pharmacy Ghent University Harelbekestraat 72 9000 Ghent Belgium Tel: +32 (0)9 264.80.78 Fax: +32 (0)9 264.81.89