RE: Anti-H. Pylori Question

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From:"Carson, Karla" <> (by way of Marvin Hanna)
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We use this procedure also and it's great!
Karla Carson HT/HTL (ASCP)
Regional Pathology Supervisor
Mercy Health Care Sacramento
e-mail <>

		-----Original Message-----
		From:	Amos Brooks []
		Sent:	Friday, January 14, 2000 7:36 PM
		To:	Sandy Julsing; histonet
		Subject:	Re: Anti-H. Pylori Question

		    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
		    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 the
		Periodic acid from the PAS stain which is a different
concentration usually. Also too much or too little Sodium Hydroxide in the
Toluidine Blue Working

		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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