From: Richard Haugland (richard.haugland@probes.com)
Date: Thu Dec 05 2002 - 18:54:17 EST
This could be of some help.
J Immunol Methods 1998 Aug 1;217(1-2):113-9 Related Articles,
<http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=PubMed&cmd=Display&dopt=pubmed_pubmed&from_uid=9776581>
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Inhibition of nonspecific binding of fluorescent-labelled antibodies
to human eosinophils.
Mahmudi-Azer S, Lacy P, Bablitz B, Moqbel R.
Pulmonary Research Group, University of Alberta, Edmonton, Canada.
Eosinophils and their products play a major role in inflammatory
reactions associated with asthma and allergic diseases. There is a
growing body of evidence that eosinophils synthesize, store, and
release bioactive cytokines and chemokines with the potential to
contribute to local inflammatory changes. Fluorescein isothiocyanate
(FITC) has been widely used as an immunofluorescent conjugate for
antibodies specific for detection of these molecules. However, FITC
is an ionic fluorochrome (negatively charged) which binds strongly
to positively charged eosinophil granule proteins. We developed new
methods to prevent charge-based interactions of ionic fluorochromes
with granule proteins, and optimised immunofluorescent staining
techniques for eosinophils. An antibody to interleukin-6 (IL-6) was
used to optimise this procedure for eosinophil-derived granule
proteins. We attempted to block nonspecific binding of FITC-labelled
anti-IL-6 using normal human IgG, foetal calf serum (FCS), bovine
serum albumin (BSA), and goat, horse, and normal human sera at
concentrations ranging between 1-10%. Only human IgG (2%; 20 mg/ml)
was able to reduce background fluorescence. These results were
confirmed using Texas Red conjugates. We also used antibodies
conjugated to a nonionic fluorochrome, BODIPY FL, to detect IL-6 in
eosinophils. Unlike FITC, BODIPY FL-conjugated antibodies did not
require strong blocking conditions (2% BSA). We recommend that a
neutral fluorochrome (BODIPY FL) should be used for
immunofluorescence studies in eosinophils. Alternatively, strong
blocking conditions may be used to decrease background binding of
FITC-conjugated antibodies.
Calman Prussin wrote:
> We are performing intracellular staining for nuclear antigens in
> eosinophils and are getting a large amount of non-specific binding. We
> have addressed autofluorescence by using APC as the fluorophore- that
> seems to work. We are using the typical aldehyde fixation (FACSLyse),
> followed by 80% methanol and then 0.5% saponin in the staining buffer.
> We have used a variety of techniques to decrease non-specific binding,
> including: variety of fixation protocols (with & w/o MeOH), titration
> of the primary and secondary Abs, using both mAb and rabbit pAb
> primary Abs, blocking with goat serum (species of secondary Ab),
> various detergents (triton, tween, OGP, NP-40) and probably other
> maneuvers I can't recall.
>
> We get good results and low non-specific binding with monocytes, but
> the eosinophils continue to evade us. Am next looking to blocking with
> human IgG (suggested concentration?) or perhaps poly-Glutamic acid (to
> block all of the basic eosinophil granule proteins).
>
> 1. Thoughts on other ways to block non-specific binding in eosinophils?
> 2. Thoughts on the use of poly-glutamic acid?
>
> Thanks,
>
> Calman
>
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