Evan's Blue & trypan blue to reduce background

From: weimer (weimer@ANTIBODIES-PROBES.com)
Date: Sat Jul 29 2000 - 02:08:43 EST


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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