RE: Indirect IF Problem (new Flow user)

Hi Tom,

Could you enlighten us a little more on the rabbit poly? It can make a
big difference if this is an affinity purified serum or just crude
hyperimmune serum.

The fact your secundary seems to bind in absence of primary is also a
cause for worry. Try another supplier, or (as a proof of concept)
directly label your primary with one of Mol Probe's Zenon anti-rabbit
reagents in a few experiments. If that pans out, you might consider
labeling your rabbit poly directly, using activated fluor (think Alexa
dyes, Phycolink preactivated PE or APC, or just biotinylate for indirect
detection in other platforms as well).

Good luck,

Guy

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Guy Hermans, PhD
Senior Scientist
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-----Original Message-----
From: Tom Higgins [mailto:thomas_higgins@med.unc.edu] 
Sent: Tuesday, April 25, 2006 8:51 PM
To: cyto-inbox
Subject: Indirect IF Problem (new Flow user)


Hello,


I was wondering if anyone can advise me on my use of an isotype control
antibody for INDIRECT immunofluorescence of PBMCs for FACS analysis
(FACSCalibur). 


I have a polyclonal IgG rabbit anti-human primary Ab that I then bind a
FITC-conjugated goat anti-rabbit secondary Ab to.  My 'isotype control'
has been to use whole molecule purified rabbit anti-human IgG.	The
problem I have is that I get a broad diffuse (over a 1 to 1.5 log scale
range) staining with the isotype control where my population of interest
is supposed to be.  My Ab of interest binds in the same area but only at
about 0.5 log scale range.  I have varied the concentrations and
incubation times. I have also tried Fc blocking using purified
non-immune goat IgG or goat normal serum, and at varying
concentrations-with no improvement. Suggestions on what I can try or
what could be causing this? 


In addition, my histograms show 3 progressively decreasing peaks all 1
log scale apart from my autofluorescence population to the 10^3 range
using my Ab of interest.  My secondary alone shows only the first 2
peaks; however, my isotype control shows 2 peaks (1 at 10^1 and the
second diffuse one at 10^3).


I would appreciate any input.  I have attached the files for reference.


Best,

Tom