RE: [Histonet] Texas Red fluorescing when slide viewed through FITCfilter

From:"Mansfield, James"

Hi Hannah,

Depending on the brightness of the fluorophores you are using, it is
quite possible for the FITC to show up in the Texas Red channel. There
is a significant (about 10% of max) FITC emission at 600 nm (the peak of
Texas Red emission).

You can plot out the spectra of various fluorophores at:

Unfortunately, there are only ways to prevent the FITC from bleeding

1. Reduce the amount of FITC on your slide so that the 10% bleed through
is not visually apparent.
2. Use a multispectral imaging approach and linear unmixing to separate
the contributions of the two fluorophores from each other.



James R. Mansfield
Product Manager, MSI Systems
Senior Spectral Imaging Scientist
35B Cabot Road
Woburn, MA, 01801
Phone: +1-781-935-9099 x 117

> -----Original Message-----
> From: 
> [] On Behalf 
> Of Hannah, Michele F.
> Sent: Wednesday, July 26, 2006 3:05 PM
> To:
> Subject: [Histonet] Texas Red fluorescing when slide viewed 
> through FITCfilter
> We are currently working on a fluorescent IHC protocol using 
> Vector Lab's Texas Red Avidin DCS and Fluorescein Avidin DCS. 
>  Eventually, the slides will be stained with both 
> fluorophores, but for now we are testing with each 
> separately.  The problem we are having is slides that are 
> stained with Texas Red will be visible using both the TX Red 
> filter and the FITC filter.  We also have the reverse 
> problem, the FITC stained slides show up with both filters.  
> The slides are kept completely separate (different stains are 
> not washed together) throughout the protocol.  We are also 
> using DAPI which is included in Vector's Mounting Reagent; 
> that is showing up perfectly.
> Does anyone have any ideas on what could be causing this?  
> Any help would be appreciated!
> Michele
> Michele F. Hannah  M.S.
> Department of Molecular Microbiology & Immunology Johns 
> Hopkins Bloomberg School of Public Health
> 615 North Wolfe Street  Room  E3201
> Baltimore, Maryland 21205
> Phone: (410) 614-7794
> Fax:     (410) 955-0105

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