Re: IHC of non-human tissue

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From:John Bliss <> (by way of histonet)
To:histonet <>
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Dear Zenobia Haffajee:

If you are using a commercial system that is designed for human tissue
(e.g., the secondary antibody does not react with human Ig) and the
secondary antibody is a polyclonal then you can spike the secondary
antibody with 3% normal serum from the same species as the tissue. This
will homogeneously absorb any reactivity with Ig from that species.
However, it will also reduce the sensitivity of the secondary antibody
which can mostly be recovered by increasing the incubation time. If you
put together your own detection system then there is a wide choice of
secondary antibodies that can be used to avoid the problem.

The more challenging situation is detecting a mouse primary antibody on
mouse tissue. If you are lucky there will not be a significant enough
amount of endogenous Ig in the tissue to cause background. Otherwise, I
would recommend Zymed's HistoMouse-SP Kit. This is the original 'mouse on
mouse' kit and it is the only kit that uses Zymed's patented BEAT(tm)
blocking system. Here are a couple of papers that cite the HistoMouse-SP
kit: Lu, Y.P. et al; Enhanced Skin Carcinogensis in Transgenic Mice with
High Expression of Glutathione Peroxidase or Both Gluthione Peroxidase
and Superoxide Dismutase. Cancer Research 57:1468-1474 (1997)
Gingrich, J.R. et al; Metastatic Prostrate Cancer in a Transgenic Mouse.
Cancer Research 56:4096-4102 (1996).

Please contact Zymed if you need more details.


John Bliss
Zymed Laboratories, Inc
458 Carlton Court
So. San Francisco, CA 94080
Tel: 800-871-4494
FAX: 650-871-4499

Ms Louise Taylor wrote:

> Hi Histonetters
> I have 2 queries ? I have always done immunohistochemistry (IHC) on
> human tissue. I understand the theory and enjoy doing IHC . I am
> however curious about the protocol when doing IHC on mouse,rabbit or
> any other host . Does one need to use a host absorbed secondary
> antibody  or is it type specific? I gather that there may be cross
> reaction from species to species. Any information on this would be
> helpful. Thanks in Advance.
> Second question: Has anyone used the CSA (Dako) kit; It is great to
> amplify minuuuute antigenic sites, however I do sometimes get a lot
> of non-specific staining. I normally when using the CSA kit , use an
> avidin/biotin block anyway. This strong punctate dot-like signal on
> the membrane of cells is on both the test as well as the negative
> control. Has anyone experienced the same problem? Should I dilute the
> primary even more or is there something else I could try? I have
> tried x-tra washings as well as Triton -X-100/BSA in my buffers.
> All help is appreciated.
> Cheers
> Zenobia Haffajee
> Anatomical Pathology
> South Africa


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