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We have addressed this question in our system, looking at intracellular
antigens in PFA fixed cells. In this system:
1. Nonspecific binding can be quite high AND often may differ between specific
antibody and isotype control
2. The frequency of positives may be quite low.
Cells to be stained are first preincubated with an excess (w50-100 mcg/ml) of
either UNLABELLED specific antibody or isotype control for 1 hour. Identical
amounts of labelled specific antibody are then added to each tube and stained
for 30 min.
This yields a negative control in which specific binding has been blocked by
an excess of unlabelled antibody, but in which nonspecific staining has been
unaffected. We have operationally used the staining of the blocked sample to
represent the nonspecific staining inherent in the system; any staining above
this level is thus by definition specific staining.
This may be a little more difficult than your average staining, but you should
be more secure in knowing that your staining is specific.
Calman Prussin
Allergic Diseases Section
National Institute of Allergy and Infectious Diseases
National Institutes of Health
(301) 496-1306
FAX (301) 480-8384
---------------------- Replied Message Body ----------------------
Date: 3-1-1995 3:34pm
From: {Dennis_Young@CIS.ucsd.edu}:unix:niaid
To: calman prussin:10:niaid
Subj: Re: Dim Fluoresscence
Also-to: cytometry@flowcyt.cyto.purdue.edu
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1) The subtraction option in Lysys II can show the channel per channel
differences in your sample.
2) Kolmogorov-Smirnov is available (It's even Russan :) (Young, IT, Journal of
Histochemistry and Cytochemistry, Vol. 25, No. 7, pp. 935-941, 1977) (NO
relation).
3) Another "quick and dirty" way is to overlay the control and set the marker
at
the intersection of the curves. With dim samples, this can be as much as 20% of
the control! (Chapter 5.2.2 , Supplement 4 Current Protocols in Immunology,
1992)
The *MOST* important problem with dim fluorescence is that you *WILL* see
differences even when they are purely due to natural variability. The binding
should confirmed by other methods.
There are also numerous ways to amplify your signal.
*****************************************************************************
* Dennis J. Young Voice : (619) 543-3928 *
* Flow Cytometry Core Facility FAX : (619) 543-7487 *
* University of California, San Diego USA e-mail: djyoung@ucsd.edu *
*****************************************************************************
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