Double labelling immunohist. with 2 mouse ab.s

chelack at admin3.usask.ca chelack at admin3.usask.ca
Thu Aug 15 11:07:03 EST 1996


Keith,
        Do you need fluorochromes on these sections?  If not then I would
recommend the following:

        1.Block Non specific sites with normal sera.

        2.Incubate primary #1.

        3.Wash

        4.Incubate secondary HRPO conjugate

        5.Wash

        6.Apply a permanent chromogen such as Di amino Benzidine, gives
        a nice Brown colourat the reaction site.

        7. Remove the first primary using either enzyme (We use Protease XIV
        0.05 mg/ml) or a dilute acid solution (I think a 0.1 N HCl for 10 min
        should do the trick)  Remember to use poly lysine or silaned treated
        slides or your sections will come off.

        8. Reblock with 5% normal sera

        9.Go through with the second primary only this time use a chromogen
        like AEC which although it is soluble in organic solvents gives a
        nice red end product. Counterstain with Hematoxylin and coverslip
        with a water based mounting media.

A couple of caveats to the above;

        It may be that one or both of your antigens do not survive the elution
        process.  If it does not work one way it may be that it might work if th        the second antigen is stained first.

        If neither one survives the elution process, and you are working with
        frozen sections you could try a brief (10 sec) "fixation" step prior
        to the elution using 0.05% paraformaldehyde which may stabilize the
        antigen.  This tends to be tricky because the immunoglobulins are also
        fixed to the section, it may be best to try the fixation prior to any
        staining, so much depends on the nature of your antigens you are trying
        to detect.  We have done this with a number of infectious agents and
        some cell surface markers on formalin fixed tissues, but it is not
        my favorite pastime.  Good Luck.

                        Regards

                        BJC - Brian

The spark of life is to accepted as a sacred trust to be transmitted
undimmed to future generations.



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