[Histonet] Texas Red fluorescing when slide viewed through FITC filter

[Hannah, Michele F.]

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