[Histonet] taupathies, LB & amyloid: IHC labeling followed by staining question

[Elizabeth Chlipala]

I find that when coupling IHC and special stains the best chromogen to use is DAB, unless the colors are too similar.  We would traditionally run the IHC first with the DAB chromogen which is not going anywhere and then essentially counterstain with whatever special stain you are performing.  Basically you run the special as you would normally after the IHC is completed.

I would not switch to a AP detection system most of those chromogens are not as permanent as DAB.  Brown IHC staining combined with the reddish orange from the congo red with a hematoxylin counterstain should look nice.  You can then dehydrate, clear  and mount in a permanent mounting media so why would you want to mount in aqueous.  I would keep it simple.

Good Luck.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Tyrone Genade
Sent: Friday, January 16, 2015 1:23 PM
To: histonet
Subject: [Histonet] taupathies, LB & amyloid: IHC labeling followed by staining question

Hello,

I found the following study, http://www.ncbi.nlm.nih.gov/pubmed/12000724,
where sections were probed with alpha-synuclein antibodies and then developed with DAB. Then the sections were stained with Congo Red for amyloid. The afore mentioned article references
http://jhc.sagepub.com/content/10/3/355 for the amyloid protocol. I assume they, the first article, had used the alkaline Congo Red method. The results look good and I can use this.

Does anyone have any experience in doing such a double labeling experiment?
Are there any problems that could be anticipated? If I use an alkaline phosphatase chromogen instead, could I expect problems? I am thinking of trying the MultiView kit by Enzo Life Sciences:
http://www.enzolifesciences.com/ADI-950-101/multiview-mouse-hrp-mouse-ap-ihc-kit/
and perhaps using some of the more vibrant chromogens:
http://www.enzolifesciences.com/platforms/immunohistochemistry/#An-Unrivaled-Selection-of-High-Definition-Chromogens
. Anyone have any references to papers that spell out a protocol? From what I can make out, the slides were first developed for IHC and then transferred to NaCl saturated 80% ethanol and then to the Congo Red where after the slides were dehydrated and cleared. (I assume, I could just as well mount in an aqueous mounting medium such as glycerol or
mowiol/n-propylgallate?)

Thanks for the answers to my previous question. Seems 10 micron is a good middle ground for the demonstration of the various pathologies from serial and double-stained sections.

Thanks
--
Tyrone Genade
Orange City, Iowa
tel: (+1) 712 230 4101
http://tgenade.freeshell.org
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