Evan's Blue (and trypan blue were used in the earlier days of direct immunofluroesence and the study of autoantibodies. Each was used on tissues/cells at 0.001% to 0.1% in water or buffer. Dip the slide with tissues/cells into the dye for 2 to 30 seconds, wash with water/buffer, coverslip with aqueous medium. They are soluable in aqueous mounting media and diffuse out quickly. Drop the coverslip, wash, restain and coverslip as many times as the tissues/cells can withstand. It does reduce background significantly; both unwanted fluroecsence due to the conjugate and autofluoresence due to structural elements in the tissues/cells. The dyes fluoresce red, abolishes the unwanted conjugate staining by energy transfer but has very little effect on desired specific staining - how do it know?? Typically, immunohistologists/cytologists use a red suppression secondary filter, BG 38, to filter out the red fluorescence of the dye and leave a nice black background to enhance the viewing of desired specific staining. I believe any early (1950-1970) good book on immunofluorescence, of which there are two, one being - Narin: 'Fluorescent Protein Traceing' and the original papers of Albert Coons - whom, as we all know received the Noble Prize for the invention of fluorescent protein traceing (immunofluorescence) will give further details. Bob Weimer "Geneva M. Omann" wrote: > Hello Flowers, > Along the lines of Shannon Ritchie's question below, I have also heard > of using trypan blue or crystal violet to quench the fluorescence of > externally bound ligands. I believe the mechanism is by energy transfer. > Has anyone else heard of this and if so do you have references for the > mechanism and protocols? > > ----- Original Message ----- > From: Shannon C. Ritchie <sritchi@emory.edu> > To: cyto-inbox > Sent: Wednesday, July 26, 2000 1:42 PM > Subject: trypan blue to reduce background > > > > > I have heard of using trypan blue to reduce background. Does anyone know > > the concentration and protocol? > >
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