If these smears are just fixed in acetone you could probably eliminate
the retrieval portion of your protocol.  I would also block for endog.
peroxidase activity.

JPaulB42@aol.com wrote:

> Hello fellow Histonetters:
>
>        We have started experimenting with Zymed's TRAcP antibody with
> poor results on peripherial smears.  Does anyone have any guidelines
> to follow to bring this antibody up and running?  Washing off is a
> problem as well.
>
> 1.  Fix in Acetone for 1 minute.
> 2.  Steam retrieval in EDTA for 30 minutes.
> 3.  Cool for 30 minutes.
> 4.  Primary antibody for 60 minutes.
> 5.  Link for 30 minutes  (LSAB2)
> 6.  Strep for 30 minutes.
> 7.  DAB for 5 minutes
> 8.  Counterstain with Hema for 5 - 8 seconds.
>
> The procedure is run on DAKO Autostainer.
>
>        Thanks everyone for your help.
>
> Jim Bradford
> ORMC
> Orlando, FL