Yep,
Definitely an issue.
You can easily stain the IPX slides with H, though the discernibility of the
cells and tissue structure with the H will depend on the degree of DAB
product laid down (eg I would expect it to be difficult with a Vimentin IPX
compared to a CD15).
(Grosset, A. A., Loayza-Vega, K., Adam-Granger, É., Birlea, M., Gilks, B.,
Nguyen, B., ... & Trudel, D. (2017). Hematoxylin and Eosin Counterstaining
Protocol for Immunohistochemistry Interpretation and Diagnosis. Applied
immunohistochemistry & molecular morphology: AIMM.)
As for doing another IPX on the existing IPX stained section (with DAB as the
chromogen), you will have to use a different label (eg alkaline phosphatase).
The result will depend on the cell compartment the two antigens exist. If the
DAB is nuclear, then a cytoplasm or cytoplasmic membrane localisation with the
Alk Phosphatase will work. If both antigens are cytoplasmic, then you will not
see co-localisation in the same cell since the DAB will prevent any antibody
binding in the same compartment.
Assuming the above is good, then since the antigen retrieval tends to reverse
over time, I would include a short retrieval before the second antibody.
Now after all that, I hope the section stays on the slide!
Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Principal Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA
-Original Message-
From: Curt via Histonet [mailto:histonet@lists.utsouthwestern.edu]
Sent: Friday, 4 January 2019 4:05 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] IHC-H HELP...
Need help before I turn a mistake into an irreparable mistake...
We have some unstained slides that were supposed to get stained with H but my
guy stained them with IHC. It's complicated, we received slides and a block,
the block was for IHC, the unstained slides were for H, he inverted the
process) The point is, now the unstained slides are stained with IHC... I know
we cannot destain the IHC but we can simply run and H over them... the real
question I have is subsequent to the H this pathologist generally likes to
see the H then order IHC on them based on what he sees (we only have these
few unstained slides, don't have blocks to recut)...
So the question is... if we've already run IHC, then followed that with and
H, can we return to run IHC on the slides again? would you want to skip any
pre-treatment, antigen retrieval
I don't see this working too well myself, if they're already stained with DAB,
that would be present on the second stain...
Thoughts?
Thanks for your help.
Curt
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