[Histonet] DAB IHC with H & E staining

[Elizabeth Chlipala]

That's correct if you do not quench endogenous peroxidase activity completely you would be able to visualize the RBC's but maybe in instances like this it might be better to have the rbc's stain a different color than the DAB they would be easier to recognize, especially if they are specifically looking at neovascularization and subtle changes in the vasculature.  DAB can be great but it also can pose problems due to endogenous pigments that appear light brown or brown in color.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
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From: David Wright [mailto:dw18 at uchicago.edu]
Sent: Wednesday, January 06, 2016 2:16 PM
To: histonet at lists.utsouthwestern.edu
Cc: Elizabeth Chlipala
Subject: RE: DAB IHC with H & E staining

Hi Histonet, Marcus and Liz

I agree with everything Liz says about precipitated DAB being wonderfully robust so that you can do a whole range of stains successfully after using it.

I am slightly puzzled that Marcus wants to reveal RBCs after a DAB reaction. Doesn't it just happen any way? Haem/hemoglobin provides a powerful non-specific peroxidase (the frothing you get with peroxide on dried blood) which will also precipitate DAB in the RBCs - I see it whenever I don't fully perfuse my brains. If you're not seeing this currently, and you are sure there are RBCs present, it might simply suffice to ease up on whatever procedure you use to "kill' endogenous peroxidases before your Ab incubations, so that the haem can go to work on the DAB.

Note that if you happen to use thick sections for floating immuno, you really don't want to use eosin. There is so much material that everything is red. (There is a similar 'too much' problem with using solochrome cyanine on thick sections for myelin...)

best to all - David
==
David A. Wright, Ph.D.
University of Chicago
Section of Neurosurgery, MC3026
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ORIGINAL MESSAGES
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Histonet Digest, Vol 146, Issue 4 Message: 7
Date: Wed, 6 Jan 2016 10:20:30 -0700
From: Elizabeth Chlipala <liz at premierlab.com<mailto:liz at premierlab.com>>
To: Marcus Green <marcus.green at oncology.ox.ac.uk<mailto:marcus.green at oncology.ox.ac.uk>>, "histonet at lists.utsouthwestern.edu<mailto:histonet at lists.utsouthwestern.edu>"
Subject: Re: [Histonet] IHC with H & E staining

Marcus

You can stain with H&E after DAB if you think it would help your how you analyze your slides.  DAB with the addition of a special stain has been published and for us depending upon the project that we are working on we may choose to use a different counterstain after an IHC that has DAB as an chromogen.  For example we have worked with macrophage markers counterstained with Prussian blue, another example is brain IHC markers counterstained Cresyl ect Violet rather than hematoxylin.  DAB is extremely stable and therefore many special stains or different counterstains can be used.

If you are running an image analysis algorithm most canned algorithms are set up to function with a hematoxylin counterstain - you would need evaluate if a different counterstain would decrease the accuracy of your algorithm but you may have the ability to design an algorithm with the counterstain of your choice.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
liz at premierlab.com<mailto:liz at premierlab.com>
________________________________________
From: Marcus Green via Histonet [histonet at lists.utsouthwestern.edu]
Sent: Wednesday, January 06, 2016 4:26 AM
To: histonet at lists.utsouthwestern.edu<mailto:histonet at lists.utsouthwestern.edu>
Subject: [Histonet] IHC with H & E staining

Dear Users,

Happy New year - I hope this finds everybody well!

I was asked a very simple question yesterday - why don't you do H&E counterstaining on DAB stained samples. The question came about as we're looking at CD31 staining for blood-vessels and some look ruptured (we're keen to see red blood cells).

Instead of doing a consecutive cuts, a CD31 stain (Haem counterstain) on one slide and H&E on another, the question was asked why not do the Eosin stain as part of the counterstain....

I've never seen it done in the literature or in the clinic, and I've never asked why it's not done. Any assistance or advice would be greatly welcome - and my sincerest apologies if this is a very basic question?

Thanks in advance for your time,

kind regards,
Marcus,
Department of Oncology - University of Oxford,