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Author Topic: adequate counterstaining for DAB  (Read 32423 times)

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Offline madeleine huey

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adequate counterstaining for DAB
« Reply #15 on: March 10, 2006, 10:37:55 PM »
I never have problem with couterstaining after DAB.  The only time fail is if Hematoxylin is expired, then the nucleus become brown also (hard to distinguish chromagen DAB vs counterstain).  
Here's what I normally do; counterstain with Sigma Hematoxylin Gill #1 for 1 min - 2 min, then rinse off blue with tap water until water become clear (appx. 1/2 min with running water), then blue nuclei with PBS for 1'-2', or until desire couterstain color (observe under microscope & write down the desire timing for future reference).  Wash off PBS with 3x distilled water, then dehydrate, clear and mount with Permount or any permanent mounting solutions.
Good Luck!
Madeleine

adequate counterstaining for DAB
« Reply #15 on: March 10, 2006, 10:37:55 PM »

Offline ImmunoNYC

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Re: Counterstaining and DAB
« Reply #16 on: March 13, 2006, 12:45:34 AM »
Hi, I am very curious about your suggestion. Can you give some details as to where you buy this and your protocol? THANKS!! :D[/color]
Quote from: "njcragg"
Just thought I would add our methods to the discussion as it is something I've realised we do differently than the majority.  As image analysis requires a significant distinction between brown and the counterstain colour, we've always used Thionine as a blue counterstain, for better contrast.  We actually use it for all IHC, not just where image anaylsis is employed, because we feel it is a better when doing manual microscopy.

Offline njcragg

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Thionine Counterstaining
« Reply #17 on: March 13, 2006, 11:55:36 AM »
Protocol
80% methanol for 10 minutes
Thionine for 6 minutes
5 dips in 95% alcohol/IMS  x2
Absolute alcohol (99% IMS) for 1 minute

We buy thionine as powder from Sigma.
ic

Thionine Counterstaining
« Reply #17 on: March 13, 2006, 11:55:36 AM »