Re: Anti-H. Pylori Question

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From:Amos Brooks <> (by way of histonet)
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    In order to not be information stingy I'm posting the Alcian Yellow
procedure to everyone.:-)

    1) Periodic Acid 1%
    2) Sodium Metabisulfite 5%
    3) Alcian Yellow 1% (filter this, its kind of gooey)
                --Poly Scientific item # s2423
    4) Toluidine Blue Stock Solution
                --0.1% Toluidine Blue (Poly Scientific #s2424)
    5) Sodium Hydroxide 3%
    6) Toluidine Blue Working Solution <--- this needs to be prepared fresh
                Toluidine Blue Stock Solution --- 0.5 ml
                Distilled Water ------------------ 50 ml
                Sodium Hydroxide -------------- 2 drops

    1) Dearaffinize and hydrate slides to distilled water
    2) Oxidize in 1% Periodic Acid for 10 min.
    3) Rinse well in tap water
    4) Place slides in Sodium Metabisulfite 5% for 5 min.
    5) Rinse in tap water for 2 min.
    6) Stain in Alcian Yellow 1% for 5 min.
    7) Rinse well in tap water
    8) Stain in freshly prepared Toluidine Blue Working Solution for 3 min.
    9) Rinse well in tap water
    10) Dehydrate QUICKLY then clear and mount as routine

    Helicobacter pylori --- Blue
    Mucin ---------------- Yellow
    Background ---------- Pale Blue

    This Alcian Yellow is yucky stuff dont forget to filter it before using
it. Whatever you use to stain the Alcian Yellow step in will be stained
forever so
choose your glassware accordingly. The bugs should have a sharp contrast of
blue on yellow epithelium you should be pleased. Common screw ups are using
Periodic acid from the PAS stain which is a different concentration
usually. Also too much or too little Sodium Hydroxide in the Toluidine Blue

good luck
Amos Brooks

Sandy Julsing wrote:

> Hi Amos,
>    Thanks for the reply.  Could you e-mail me the alcian yellow
>procedure?  Or fax it to 1-920-738-6438 "Attention Sandy in Histo"
>Thanks much!    Sandy
> >>> Amos Brooks <> 01/12/00 02:15PM >>>
> Hello,
>     Our lab does a routine HPY stain on GI biopsies (Alcian Yellow). The
>doctors then ask for HPY-IHC on
> suspicious cases. (Obvious positive cases do not need the extra charge)
>We antigen retrieve the sections by
> steaming them in citrate buffer for 20 min. We then incubate for 30 min
>(room temp.) using DAKO primary
> antibodt at 1:100. Then Envision + as a detection kit.
>     There seems to be far less background staining with this detection
>kit than with LSAB+ but the kit costs
> more.
> Amos Brooks
> Sandy Julsing wrote:
> > Hello Histonetters,
> >    I have two questions re: anti H.Pylori.
> > ~We are using DAKO's antibody at 1:400 15 minutes
> > ~10 min AR in steamer and citrate buffer pH 5.8
> > ~LSAB+ 15 min, 15 min, 5 min
> >
> > My questions are...
> >
> > 1-How far out have others diluted the anti-H Pylori and using which
>antigen retrieval and detection kit?
> >
> > 2-Do you do a rapid Giemsa or Diff-Quik first and use  the anti
>H.Pylori only if it is negative?
> >
> > Thanks a bunch - Sandosis

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