[Histonet] Bone adhesion problems after HIER
anh2006 <@t> med.cornell.edu
Thu Jan 11 16:02:21 CST 2007
I think the IHC problems only arose b/c Patrick
dropped the temp of retrieval down to 70 deg C. I
can confirm from personal experience that for
most antigens it doesn't work. Even 80 deg C (the
point at which the sections start to come off
Plus slides) is largely ineffective. 95-99 deg C
is critical retrieval temp in my experience.
I am going to try those slides recommended by
Nancy, maybe Patrick can do the same (or your
Elmer's method which for me isn't an option as we
don't cut our own sections anymore so cannot
control those parameters too easily).
> I am somewhat confused about your e-mail,
>because it is titled "bone adhesion problems"
>and at the end it seems that you solved that
>problem, but are having problems with the IHC
> 1-for the adhesion: I always used 1 mL of
>Elmer's glue in a regular 2L water bath and
>"fished" the sections with superfrost + slides
>(for both nails and bones). Did not lose
>sections during HIER.
> 2-about the failure with IHC I cannot try to
>help you if I do not know what epitope your are
> René J.
>"Leung, Patrick" <patrick_leung <@t> merck.com> wrote:
>I am having problems with cortical bone detaching from the slide during
>the antigen retrieval step. The samples I am staining are paraffin
>embedded mouse tibia.
>I am currently using Superfrost+ slides. The AR is with citrate buffer
>(pH6.1) from Dako performed in a waterbath for 15 minutes. The slides
>are baked overnight at 59C prior to deparaffinizing.
>I have tried using a steamer and microwave, however I still ended up
>with the same results. In addition Superfrost GOLD slides did not help
>in keeping the bone on. I found that performing HIER at 70C for three
>hrs as suggested in the archives to be ineffective in exposing my
>antigen (although the bone did stay attached).
>Do you have any suggestions?
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