[Histonet] Tyramide

[Adam .]

I've had good luck with the biotinyl tyramide system from Perkin-Elmer

Here is what I do using an unlabeled primary antibody
1) Block endogenous peroxide since you use peroxidase chemistry for this to
work
2) Block avidin
3) Block biotin
4) Block with TNB buffer (the kit's equivalent of blocking serum)
5) Add primary antibody
6) Add biotinylated secondary antibody
7) Add SA-HRP (comes with kit)
8) Add biotinyl tyramide reagent. The HRP will deposit biotinylated tyramide
near your antigen.
9) Add SA-HRP
10) Add DAB

It works well. It also works if you replace step 9 with SA-fluorophore and
don't use DAB at all.

If your primary antibody is already labeled with HRP, you could do try this:

1) Block endogenous peroxide since you use peroxidase chemistry for this to
work
2) Block avidin
3) Block biotin
4) Block with TNB buffer (the kit's equivalent of blocking serum)
5) Add primary HRP conjugated antibody
6) Add biotinyl tyramide reagent
7) Add SA-HRP
8) Add DAB

I've never tried it, but I'd expect it won't amplify as well as my method.
However, I see no reason why it shouldn't work.

Good luck,
Adam

On Tue, Sep 28, 2010 at 11:13 AM, <sgoebel <@t> xbiotech.com> wrote:

>
>   Hello  world...So  trying  to  amplify my signal using tyramide.  ; I
>   bought  a  kit  from  Invitrogen  to  use  with  fluorescence  and the
>   fluoresce  nce didn't work at all!  The PHD that is helping with this
>   said  that  h  e  thought  we  should try it using DAB instead of the
>   fluor.   I  called  t  he  Invitrogen technical help...WOW!!!  I have
>   never  had  my  own questio n repeated back to me so many times, that
>   guy  was  a  moron on IHC, rude, and should be fired!!!  So I come to
>   ya'll!!   Any  idea  where  I can b uy tyramide to be used with a DAB
>   developer?   My  primary  antibody is a lready conjugated with HRP, I
>   just  want  to  amplify  with  with  the  tyramide  a  nd  stain with
>   DAB/hematoxylin.  Any help would be awesome!!!
>
>
>   Sarah Goebel, B.A., HT (ASCP)
>
>
>
>   Histotechnician
>
>   < i>X Biotech USA Inc.
>
>   8201 East Riverside Dr. Bldg 4 Suite 100
>
>   Austin, Texas& nbsp; 78744
>
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