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Author Topic: Which is the best buffer for DAB?  (Read 4052 times)

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Offline F.Bernal

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Which is the best buffer for DAB?
« on: June 26, 2006, 06:24:01 AM »
Hello all,

This may sound a bit a stupid, too-basic question :oops: but, which is the best buffer for DAB staining? I would prefer to use PBS for the reaction, as this is the buffer I use for washing during the IHC and it is more coherent, but changing protocols is something I am scared of. Tradicionally we have always used TBS, but I have met people that uses Tris without salt, other that says PBS works perfectly well and, the most annoying, each uses a different pH, which I also find in the datasheet of the DAB tablets I use (Dako), where for Tris (no salt) choose a pH 7.6 and for PBS a pH 7.0.

I know that pH may be critical for a good staining, which is the best range? As I undertand NaCl is not mandatory, isn't it?

Thanks!

Which is the best buffer for DAB?
« on: June 26, 2006, 06:24:01 AM »

Offline richard03

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Which is the best buffer for DAB?
« Reply #1 on: June 26, 2006, 10:32:22 PM »
I vote for PBS (pH7.2), and it is slightly better than TBS (pH7.6) as I did side by side comparation before.

Richard

Offline neusci

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Which is the best buffer for DAB?
« Reply #2 on: August 22, 2006, 02:46:12 PM »
Sorry to toss another at you, but I actually just use dH20.

Offline F.Bernal

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Which is the best buffer for DAB?
« Reply #3 on: August 23, 2006, 04:11:55 AM »
:shock: I am amazed it actually works, as peroxidase reaction is heavely dependent on pH. I would recomend you to use some kind of buffer, at least to have consistent results, as in water pH may fluctuate considerabely.

Continuing with the topic, I finally also did a paralel assay with PBS pH 7.2 and Tris buffer pH 7.6 (no saline!). Comparing similar incubation times, while TB induced a more intense reaction, it also had slightly higher background. So I believe the buffer may be selected depending on the abundancy of the antigen you are working with. Personally I will continue with TB, just for internal coherence.

Offline ImmunoNYC

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Re: Which is the best buffer for DAB?
« Reply #4 on: August 24, 2006, 01:36:13 AM »
I use the buffer that comes with the DAB kits I use. I will check what it is and get back to you. Probably PBS based.[/color]

Re: Which is the best buffer for DAB?
« Reply #4 on: August 24, 2006, 01:36:13 AM »