We are experiencing exactly the same problem with our ISH work. We are
using a Cambio probe and the Vectastain elite ABC for detection. Most of
the nuclei stain bron with the DAB. Neither Cambio nor Vecta can offer
a solution. I have increased my peroxide incubation, added Tween and BSA
to all solutions, incubated in Horse serum and/or milk powder but I
still cannot get rid of the nuclear staining.
I am going on leave today till 6 Jan and would appreciate it if you will
pass on any suggestions.
Coskran, Timothy M wrote:
>Has anyone ever encountered the following type of IHC background problem:
>With two separate antibodies, one a cytoplasmic marker and the other a
>membrane marker, every single nucleus in the section turns brown. The stain
>itself works, as most of the time you can read through the background, but
>all test slides and negative controls (omission of primary) are affected.
>Both antibodies are rabbit polyclonals, both are steam retrieved one with
>citrate pH 6 the other with EDTA. Both are detected using a whole IgG
>biotinylated goat anti rabbit secondary followed by Elite ABC and Dako
>Liquid DAB+. Blocks included 3.0% hydrogen peroxide (aq), Dako Biotin
>Blocking system and Dako Protein block. All were stained on an IHC stainer.
>In addition, both of theses antibodies have been reliable for months (using
>the above protocol steps), until now......
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Dave Lizamore (firstname.lastname@example.org)
Faculty of Health Sciences School of Anatomical Sciences
University of the Witwatersrand
Tel +27 (011) 717 2716 Fax: +27 (011) 717 2702
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