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Author Topic: Removal of DAB precipitate  (Read 15869 times)

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Offline rosa

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Removal of DAB precipitate
« on: April 22, 2006, 01:40:31 AM »
Hi. I am new in this forum and in the HIC.
I have a lot of problems with DAB staining. I use Sigma Fast: if I do not filtrate the solution, the staining is very strong (a few minutes) with precipitate (or spots of DAB); if I filtrate it, the staining is weak (about one hour or more) and in some cases I have precipitates too.
So, I have decided to change the DAB but I ask you if you know a method to remove the DAB or to clean better the tissue sections to eliminate the excess of DAB. I can reuse a tissue section for another primary antibody incubation?
Thank you.

Rosa
Italy

Removal of DAB precipitate
« on: April 22, 2006, 01:40:31 AM »

Offline excalibur

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Removal of DAB precipitate
« Reply #1 on: April 22, 2006, 10:12:13 AM »
Sorry Rosa, DAB is permanent and cannot be removed without tissue damage. I suggest trying another companies DAB or using AEC.

Your filter may also be too fine, trying using a less restrictive filter size.

DAB should work in less than 10 minutes.
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline rosa

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Removal of DAB precipitate
« Reply #2 on: April 22, 2006, 10:51:45 AM »
Hello Paula.
Thanks for the reply. I have used a 0.22 um syringe filter and a lot of DAB solution remained in the filter. Now I have a DAB of another company (a gift of a friend of mine) and I will try it. I hope to get a good result.

Rosa

Offline ImmunoNYC

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Removal of DAB precipitate
« Reply #3 on: April 24, 2006, 12:13:35 AM »
Invest in a DAB+ kit from either Zymed or DAKO. You will never have this problem EVER AGAIN.[/color]

Offline chandika

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Removal of DAB precipitate
« Reply #4 on: April 24, 2006, 01:48:23 AM »
There could be several reasons why you get this strong DAB staining with precipitate.
1) You must use some method to block endogenous peroxidases; This can be done with H2O2 in Methanol (protocols are available in this website)
2) There could be endogenous biotin activity producing excess staining.
3) You must use better washing steps for DAB as well as the antibody systems. When antibodies are not washed properly, they trap in tissues and may give falls positive results. I generally use 3 washings in TBST each lasting 5 minutes.
CW

Offline rosa

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Removal of DAB precipitate
« Reply #5 on: April 24, 2006, 04:18:41 AM »
1) I use a TBS + H2O2 method to block peroxidase.
I have not an excess of staining but spots of DAB (like dirtiness). I try to explain: my slide is not stained (because my antibody doesn't work well) and on the tissue I see brown spots (not linked to antibody).
2) I do not know about biotin activity in my tissue
3) I wash the slides with TBS 2x5 minutes. I may increase the numbers of washings.
Today I have done again ethanol and xilene washings. Likely they were old and dirt.

Offline chandika

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Removal of DAB precipitate
« Reply #6 on: April 24, 2006, 11:21:01 PM »
Use distilled water to wash DAB (Double distilled water is better). Make it 3 washes, each for 5 minutes. How do you wash the slides ? Is it only by dipping into the jar? I use magnet stirer inside the jar so that there is good mixing.
CW

Offline discman

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Removal of DAB precipitate
« Reply #7 on: April 25, 2006, 04:37:58 AM »
Hi rosa,

I'm in the same boat... I also tried Sigma Fast and was not happy with it. I also experienced excessive staining and an increase in background compared to other DAB reagent I was using before. Finally, I went back to the DAB I used before and the stainings were fine again.

GŁnther

Offline rosa

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Removal of DAB precipitate
« Reply #8 on: April 26, 2006, 03:19:01 AM »
I use TBS to wash the slides after DAB staining. Do you think that ddH2O is better? I wash the slides on the shaker.
Tomorrow I'll try the new DAB and your suggestions. You will know the results.

Rosa
Italy

Offline ImmunoNYC

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Removal of DAB precipitate
« Reply #9 on: April 26, 2006, 11:28:20 PM »
Without a doubt for several reasons but mainly that the dH20 will quench the reaction and also if you use buffers with varying pH you may see some color differences in the DAB. For consistency always used dH20. Two x 5 minute washes is more than sufficient.
[/color]
Quote from: "rosa"
...  Do you think that ddH2O is better? ...

Offline discman

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Removal of DAB precipitate
« Reply #10 on: April 27, 2006, 03:24:27 AM »
Quote from: "MaximinaNYC"
Without a doubt for several reasons but mainly that the dH20 will quench the reaction and also if you use buffers with varying pH you may see some color differences in the DAB. For consistency always used dH20. Two x 5 minute washes is more than sufficient.
[/color]


Hi Maximina,

isn't this a bit of a contradiction? For more consistency I would rather use a buffer than water... Water might vary a lot depending on the source, whereas a buffer should be pretty well defined and reproducible.

Or do I missunderstand something in your post?

GŁnther

Offline excalibur

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Removal of DAB precipitate
« Reply #11 on: April 27, 2006, 09:55:19 AM »
Every protocol I have seen on company data sheets says to wash with distilled water after DAB and that is what I use after every chromagen I use too.
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline discman

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Removal of DAB precipitate
« Reply #12 on: April 27, 2006, 10:02:27 AM »
I know and I agree...

I don't want to be obstinate here, but I don't think the 'consistency' argument for water holds true...

BTW, I tried both PBS and water and did not find any differences

GŁnther

Offline rosa

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Removal of DAB precipitate
« Reply #13 on: April 28, 2006, 08:42:33 AM »
Hi.
Today I had a good result with the new DAB. I washed with H2O 2x5 min. The slides are very clean. Likely, the real problem was the DAB.

Good weekend.

Rosa

Offline ImmunoNYC

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Removal of DAB precipitate
« Reply #14 on: April 29, 2006, 12:24:07 AM »
Quote from: "discman"
.... I don't want to be obstinate here, but I don't think the 'consistency' argument for water holds true ... ...


I didn't say tap water, then you might have a point .... My Millipore water is about the most consistent thing in my lab. If I can't count on that then what in the world am I doing in a lab :P[/color]

Quote from: "discman"
BTW, I tried both PBS and water and did not find any differences


I don't mean to imply that using PBS will ruin an experiment, like you have pointed out, it may make no difference at all. But on the other hand, it may. I have had experience particularly with my favorite DAB kit from DAKO that when I use buffers of varying pH contained various salts my DAB turns different shades of brown. When doing quantitation using automated machines as I do, subtle color differences can render your automated protocols meaningless. Hence my need for consistency from everything from shade of DAB to the amount of time in hematoxylin and bluing reagent etc.[/color]

Quote from: "discman"
.... whereas a buffer should be pretty well defined and reproducible.


You might be surprised how different even PBS is from lab to lab. Different salt formulations and different pH depending on the competancy of the person making it.

I know I am probably a maniac when it comes to details like this but in my job I have found that it really counts.

Plus PBS is more expensive so if I can use water, then why not?

Finally the main reason I use it is to quench the reaction by washing the substrate away with excess product. I guess the main thing though practically speaking is just to quench well before moving on to your next step .... if PBS works for you, then go for it. It is not incorrect, just not what I, in my experience, would recommend.
[/color] :wink:

Removal of DAB precipitate
« Reply #14 on: April 29, 2006, 12:24:07 AM »