Author Topic: DAB staining issue  (Read 3736 times)

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Offline CJonathan

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DAB staining issue
« on: June 12, 2013, 04:21:34 PM »
I experimenting some issues with the 3,3' Diaminobenzidine (DAB).
First, the context: I inject Biodextran amine (BDA) tracers in some brain region, this tracers is tag with biotin so the coloration with DAB should be simple......but it's not....

Problem: When I stain on free floating section of mice brain (40m), I don't have the brown coloration usually obtained with DAB my tissue are crystal clear. I see the spot of interest in brown but all the rest of the tissue is clear. I used a lot DAB before and I'd never had that; usually the tissue is brown and the spot of interest are more black....
Here it's very difficult to see the brain structures, and I don't understand why? I changed all the product for brand new, but it still there. I am using a very simple protocol.

The protocol used:
-   3 x 5 min  wash in PBS 1X
-   0.3% H2O2 (dilution in PBS 1X) for 30min @ Room temperature
-   3 x 5 min in PBS 1X
-   ABC Kit (4 drops of A + 4 drops of B in 20ml of PBS 1X) for 1 hour @ RT
-   3 x 5 min in PBS 1X
-   15 min in DAB (0.05% ~ 2 tabs in 40ml of PBS 1X)
-   3 x 5 min wash in PBS 1X
-   Bath of alcohol for dehydrate: 50%, 75%, 95%, 100% alcohol bath. Then Xylene and Paramount.
-   Let the slides rest for 2 days.

Do you have any idea?
Thanks

DAB staining issue
« on: June 12, 2013, 04:21:34 PM »

Offline gula

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Re: DAB staining issue
« Reply #1 on: June 13, 2013, 12:55:51 PM »
Do I understand right? Your specific staining is brown, but you want unspecific background for a better orientation?

Wouldn't it be better to counterstain with hematoxylin for tissue-organization?

gula

Re: DAB staining issue
« Reply #1 on: June 13, 2013, 12:55:51 PM »