Gudrun,
Teri Johnson [TJJ@Stowers-Institute.org]gave an excellent summary of
this problem on the IHC resource groups listserver (Wednesday, 21 May
2008) and hopefully with her leave, I have reproduced it below:
"I did some rooting around in the literature and found out why you get
antibody staining (in negative control material) of mast cells, and what
you can do about it.
Summary During investigations of murine and human mast cell
immunoreactivity with potential anti-interleukin-4 antibodies,
non-specific, non-immunological labelling of mouse and human mast cells
became apparent. Non-specific, non-immunological labelling was
identified by (i) immunolabelling of mast cells when using control
isotype primary antibodies, (ii) ability of conjugated secondary
antibodies to label mast cells without prior mast cell exposure to a
primary antibody, (iii) extinction of the non-specific labelling and
retention of specific labelling when the pH of the diluting and washing
buffers is shifted from pH 7.2 to pH 6.0, and (iv) reduction/extinction
of the labelling when the antibodies are pre-incubated with soluble
heparin prior to immunostaining. The site of the reactivity on the
electron microscope level was shown to be confined to the mast cell
secretory granules. The results of this study support the hypothesis
that non-specific labelling of mast cells results from an ionic
interaction between the F(ab)2 segments of antibodies and the heparin
constituent of the mast cell secretory granules. This study points out
the necessity of stringent controls when using immunohistochemistry to
determine mast cell reactivity to various antibodies.
(Ref: Histochemical Journal, Vol. 25(9), Sept 1993, Schiltz et al)
An additional source for this (1989-1990 publication):
It is postulated that the granules of TMC bind certain antibodies by a
cation-exchange mechanism involving ionic interactions with positively
charged groups in the F(ab')2 and/or Fc segments. (J Histochem Cytochem
38:859-867, 1990)
And, finally, I found this little gem:
To avoid nonspecific binding of the antibodies to the heparin contained
in mast cells, the specificity of immunostaining was checked. PBS for
washing and dilution of the antibodies was acidified to pH 6.0.
Preadsorption of the STG I antibody with 1000 U/ml heparin (Sigma; St
Louis, MO) for 60 min at RT before use was carried out to avoid
nonspecific binding of immunoglobulins and heparin. The specificity of
the immunohistochemistry was confirmed with negative controls: absence
of primary or secondary antibody and avidin-labeled peroxidase. Normal
non-immune mouse serum was also used instead of the primary antibody.
(Ref: JHistochemCytochem 49(3): 341, Kumura et al)
Tammy: You never mentioned if you were getting mast cell staining in
your negative control. If no, then the above may not pertain to you and
you may have an issue of specific cross-reactivity with the antibody."
Good luck!
Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110
Thanks Terri,
Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
The Children's Hospital at Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318
-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Gudrun
Lang
Sent: Friday, 18 July 2008 2:31 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] hcv and mastcells
Hi all!
I did an immunohistochemistry for heptatitis C virus on skin of a man,
who formerly had an hcv infection. The mastcells showed a granular
staining in the cytoplasm. Has anyone experience or explanations with
this phenomen? Could it be due to a cross-reaction or are there really
viruses (at least
particel) in the mastcell-granula?
Thanks in advance
Gudrun Lang
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