7 replies to this topic

[miraclestrain]

Hi all,

I am preparing a stain, DAB for my immunohistochemistry study, I am trying to dissolve 0.05mgDAB in 1ml of PBS.
I followed the follwoing steps;
1. took 25mg of DAB dissovled suspended it in 100% ehtanol, warmed the ethanol to 60 deg C.
2. Then crushed it and warmed again for 1 hour,
3. Then mixed it with 1xPBS to required quantity.

DAB is not dissolving in warm ethanol nor later in PBS,
Should i warm it for a longer time or what should I do.

Thanks
MS

[HomeBrew]

Never used the stuff myself, but in the protocol here, they don't use any ethanol, or heat. They are preparing a 1% (20X) aqueous solution, and have to acidify it to get it to dissolve.

[miraclestrain]

Hey thanks for that, 80% got dissolved I am waiting for the other part to dissolve. Thanks anyways
MS

[pesji]

miraclestrain, on Aug 20 2009, 07:10 PM, said:

Hi all,

I am preparing a stain, DAB for my immunohistochemistry study, I am trying to dissolve 0.05mgDAB in 1ml of PBS.
I followed the follwoing steps;
1. took 25mg of DAB dissovled suspended it in 100% ehtanol, warmed the ethanol to 60 deg C.
2. Then crushed it and warmed again for 1 hour,
3. Then mixed it with 1xPBS to required quantity.

DAB is not dissolving in warm ethanol nor later in PBS,
Should i warm it for a longer time or what should I do.

Thanks
MS

Taken from SIGMA recommandations !

Quote

Buffer pH
will also affect solubility. High pH will deprotonate
DAB to its free base, which is not water-soluble.

[HomeBrew]

Merged the two threads by miraclestrain on this topic.

[miraclestrain]

HomeBrew, on Aug 21 2009, 10:00 PM, said:

Merged the two threads by miraclestrain on this topic.

I am not quite clear with the pH, High pH would deprotanate the molecule, i believe thats what you have mentioned. So what am I supposed to do?

I have to maintain the pH at 7.4 because I have to stain the sample.

MS

[Dr Teeth]

miraclestrain, on Aug 26 2009, 11:33 AM, said:

HomeBrew, on Aug 21 2009, 10:00 PM, said:

Merged the two threads by miraclestrain on this topic.

I am not quite clear with the pH, High pH would deprotanate the molecule, i believe thats what you have mentioned. So what am I supposed to do?

I have to maintain the pH at 7.4 because I have to stain the sample.

MS

Dissolve at a more acidic pH, then return the base to 7.4 with NaOH.

[fluffybunny]

Dear MS,

Are you still having trouble with dissolving DAB? I use DAB for my IHC study too. However, I bought a dako envision kit which comes with the secondary antibody, the DAB and the buffer as well. Ratio of DAB to buffer is 20 : 1000. Buffer recipe is not enclosed I suppose, but Maybe you can buy the kit? The Dako cat number is K5007. See if Dako just sells the DAB alone.

I'm attaching the EnVision kit's manual in case you'll like to see it.

Good luck!

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