RE: [Histonet] Re: Fluorophore adsorption emission and spectralseparation

From:"Sally Prouty"


If your just using 1 antibody and dapi. I would suggest using Alexa555, 
Texas red or Cy3 depending on your filters. You shouldn't get as much 
autofluorescence. Also cameras tend to see red better then blues and greens 
and you get a better signal to noise ratio with red dyes.


Sally J. Prouty
Research Assistant
Howard Hughes Medical Institute/University of Iowa
Department of Physiology and Biophysics
Kevin Campbell Laboratory
4283 Carver Biomedical Research Building
Iowa City, Iowa 52242
Fax: 319-335-6957

>From: "Melissa Gonzalez" 
>Subject: [Histonet] Re: Fluorophore adsorption emission and 
>Date: Mon, 24 Jul 2006 12:03:36 -0700
>I've never had much luck visualizing the blue-range emitting dyes. In my
>experience, they tend to be very weak and photobleach very easily, which
>are sentiments expressed by others I've conferred with as well. AF488 is
>usually pretty distinct, so if you are having trouble with this, I would
>think AF405 or AF35 would be much worse. DAPI is a very strong nucleic
>dye, and unless your filters are shot, should always look really good.
>If you have the filters: AF555 (TRITC) or AF594 (Texas red equivalent)
>are also very bright.
>Also, I am not sure how thick of sections you are using, but brain
>tissue structures always pop up a little nicer when you use thicker
>sections with fluorescence. Sometimes what may appear wimpy on a thin
>section will stand out on a thicker one.
>Good luck,
>-----Original Message-----
>[] On Behalf Of
>Sent: Monday, July 24, 2006 10:41 AM
>Subject: Histonet Digest, Vol 32, Issue 24
>I had an immunofluorescence question that I'd love to get some input on
>from anyone out there.  I'm trying to colocalize two antigens in rodent
>brain sections.  One of the antigens is yielding fairly weak staining-
>it also happens to be the one that I'm visualizing with Alexa Fluor 488,
>so I'm getting fairly high background autofluorescence- and I think that
>the staining could be improved by reducing the autofluorescence.  My
>DAPI staining (ex/em: 350/470) is only stains nuclei with
>minimal autofluorescence.  They offer Alexa Fluor 405 streptavidin which
>has a similar excit/emission range, so I thought labeling with the Alexa
>Fluor 405 would produce a similarly "clean" background as my DAPI
>labeling.  Also, I already have the filter sets to work with Alexa Fluor
>405 (as opposed to going to the other end of the spectrum).
>   Has anyone used Alexa Fluor 405?  What is your impression of tissue
>autofluorescence in that range, photostability/bleaching of the
>compounds, comparison of signal using other fluorophores, etc.
>   Any input would be greatly appreciated.
>   Thanks,
>   Adam Perry
>   Department of Neuroscience
>   University of Illinois at Chicago
>   Chicago, IL 60612
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