[Histonet] Re: Fluorophore adsorption emission and spectral separation

[Melissa Gonzalez]

Adam, 
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, 
Melissa


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
histonet-request <@t> lists.utsouthwestern.edu
Sent: Monday, July 24, 2006 10:41 AM
To: histonet <@t> lists.utsouthwestern.edu
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 great...it 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