[Histonet] Efficacity of fluorochrome in Immunofluorescence

[Kim Merriam]

You are likely getting some cross-reactivity with your secondary antibodies (one of them may be crossing to the other primary).  We always run a battery of control slides when validating a double-IF stain.  Run these controls and you will likely figure out where the problem is:
  
Slide Set Antibodies 
1 Primary #1/Secondary#1 
2 Primary#2/Secondary #2 
3 Isotype#1/Secondary #1 
4 Isotype #2/Secondary #2 
5 Primary #1/Primary #2/Secondary #1/Secondary#2 
6 Isotype #1/Isotype #2/Secondary#1/Secondary #2 
7 Primary #1/Isotype #2/Secondary #1/Secondary#2 
8 Isotype #1/Primary#2/Secondary #1/Secondary #2 
9 NoPrimary (diluentonly)/Secondary #1 
10 No Primary(diluentonly)/Secondary #2 
11 No Primary (diluentonly)/Secondary #1/Secondary #2 
12 Diluent only (or nothing) 
13 DAPI only 
 
Best of luck!
Kim,
Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA


________________________________
From: Natalia Fernandez <nunut86 <@t> hotmail.com>
To: histonet <@t> lists.utsouthwestern.edu 
Sent: Tuesday, January 24, 2012 4:45 AM
Subject: [Histonet] Efficacity of fluorochrome in Immunofluorescence


Hi everybody! 

Maybe you can help me.

I'm trying to do immunofluorescences to see the colocalisation between two proteins (PSD + Oligomeres) using Oregon Green (goat anti mouse) and Texas Red (goat anti rabbit). 
But my probleme, is that the analysis of the slides reveals too much colocalisation. I mean, all seems yellow, as if both fluorochromes show the same thing.

It's the first time I do fluorescence, so I don't know where could come the problem. 
I tried to do a simple staining, 1 slide with my first antibody and its fluorochrome, and the second slide with the other first antibody with its fluorochome and then check the result with the filter red and green. But it's strange because I see both color as if I had put both fluorochromes (second antibodies).

Has somebody already had this problem?
What can I do to resolve it?

Thank you very much for your help.
Natalia                         _______________________________________________
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