[Histonet] Texas Red fluorescing when slide viewed through FITCfilter

[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:

http://probes.invitrogen.com/resources/spectraviewer/

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

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.

Regards,

-Jim



--
James R. Mansfield
Product Manager, MSI Systems
Senior Spectral Imaging Scientist
CRi
35B Cabot Road
Woburn, MA, 01801
 
Email: jmansfield <@t> cri-inc.com
Web: www.cri-inc.com
Phone: +1-781-935-9099 x 117
 
 

> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu 
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf 
> Of Hannah, Michele F.
> Sent: Wednesday, July 26, 2006 3:05 PM
> To: histonet <@t> lists.utsouthwestern.edu
> 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
>  
>  
>