Re: mouse monoclonal on mouse tissues. 2 methods.

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From:"J. A. Kiernan" <>
To:Daniel Jimenez <>
Date:Thu, 27 May 1999 12:15:32 -0400 (EDT)
Content-Type:TEXT/PLAIN; charset=US-ASCII

On Wed, 26 May 1999, Daniel Jimenez wrote:

> I am currently using a mouse primary antibodies in mouse tissues, but I
> have high background probably due to the inability of the
> anti-mouse-secondary antibody to distinguish between the primary antibody
> and the endogenous mouse Ig in the tissue. 
> Does anybody know how to eliminate the backgroung problem without changing
> the antibody? 

  There are two strategies.
  A. (Hierck +3 1994 J Histochem Cytochem 42:1499-1502)
    1. Incubate the diluted monoclonal with HRP-labelled
       rabbit anti-(mouse serum) overnight in fridge.
       This joins all the monoclonal antibody molecules to 
       labelled rabbit 2ndary.
    2. Add normal mouse serum to the mixture. Wait 2h, 4C.
       This blocks the left-over rabbit 2ndary antibody 
    3. Apply the mixture to the sections for 4h.
       The Fab segments of the monoclonal antibody molecules
       will attach to specific antigens in the tissue, with
       their already bound, labelled rabbit 2ndary. The
       other HRP-labelled rabbit antibody molecules don't
       bind because they are already gummed up with 
       immunoglobulins from the normal mouse serum.
    4. 3 rinses
    5. Histochemical detection of HRP. 

  B. (Lu & Partridge 1998. J Histochem Cytochem 46: 977-983)

    1. Pre-treat the sections with a mixture of Fab and Fc
       fragments, made by incubating an anti-(mouse immunoglobulin)
       with the proteolytic enzyme papain. The Fab fragments
       will attach to mouse immunoglobulin in the section. 
    2. Apply the mouse monoclonal primary antibody.
    3. Apply a labelled anti-(mouse immunoglobulin). This
       joins on to the bound monoclonal, but not to
       endogenous mouse immunoglobulin, which is blocked by
       the unlabelled Fab fragments applied in Step 1.
    4. Detect the label.

  For details, you'll need to consult the papers mentioned.
  One of our graduate students here had good results with
  method A about 3 years ago. I don't know anyone who's
  tried B. Method B looks simpler to do, especially if
  you can buy the papain-digested anti-(mouse Ig) concoction

  Hope this helps,

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1

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