Hi, I am trying to prepare DAB for IHC. I have recently purchased 5g of DAB http://www.sigmaaldrich.com/catalog/product/sigma/d5637?lang=en®ion=AE and according to our protocol, I need to dissolved 1g of DAB in 40 ml of distilled water and aliquot into 40 vials.
Unfortunately the DAB does not seem to dissolve. I have tried adding HCL but it didn't work.
I then decided to aliquot the solution into vials, but because of the high amount of undissolved DAB, I got DAB powder stuck in the tips (that is when I decided to filter the solution).
Filtering produced a clear yellow colored solution. I have in the past received one vial of prepared DAB from another lab and the solution was not clear and yellow! It was cloudy and silver in color.
Help!!!!
Dear Elena, The DAB specification sheet from Sigma which Bolok send you explains well why you had a problem. You were trying to make a very high concentration of DAB (1g in 40ml = 1000 mg/40 ml = 25 mg/ml). The most often used concentration when working with DAB in substantia (in powdered form) is, as mentioned in the Sigma spec sheet is 0.66 mg/ml (or 10 mg in 15ml of TBS buffer). Your concentration was 40 times stronger. If you wished to have a concentrated stock - there is a better way of preparing DAB for future experiments, making a 'ready to use' aliquots. Long time ago I have used to prepare in my lab 50 ml of DAB in TBS buffer and keep it frozen 1 and 5 ml aliquots at -20degC. The solution worked well after defrosting when needed.
There is also an alternative. Using DAB in powder for is very cost-effective, but discouraged because of health and safety reasons. I use now, and recommend to students, another DAB preparation from Sigma - DAB tablets which are paired with buffer tablets. There are tablets (SIGMAFAST) available for making 1, 5 and 15 ml of dH2O,no need for buffer. This saves you from the need to weigh DAB powder, you dissolve the tablets just before end of your experiment and add a specified amount of H2O2. Good luck with your experiments.
Tablets for 5 ml:
http://www.sigmaaldrich.com/catalog/product/sigma/d4293?lang=en®ion=GB
It needs to mix, using a stirrer and magnetic flea for a long time, therefore cover so not exposed to the air. Clear or straw coloured is good.....Black or brown colour is bad..means it has already oxidised
Dear Elena, The DAB specification sheet from Sigma which Bolok send you explains well why you had a problem. You were trying to make a very high concentration of DAB (1g in 40ml = 1000 mg/40 ml = 25 mg/ml). The most often used concentration when working with DAB in substantia (in powdered form) is, as mentioned in the Sigma spec sheet is 0.66 mg/ml (or 10 mg in 15ml of TBS buffer). Your concentration was 40 times stronger. If you wished to have a concentrated stock - there is a better way of preparing DAB for future experiments, making a 'ready to use' aliquots. Long time ago I have used to prepare in my lab 50 ml of DAB in TBS buffer and keep it frozen 1 and 5 ml aliquots at -20degC. The solution worked well after defrosting when needed.
There is also an alternative. Using DAB in powder for is very cost-effective, but discouraged because of health and safety reasons. I use now, and recommend to students, another DAB preparation from Sigma - DAB tablets which are paired with buffer tablets. There are tablets (SIGMAFAST) available for making 1, 5 and 15 ml of dH2O,no need for buffer. This saves you from the need to weigh DAB powder, you dissolve the tablets just before end of your experiment and add a specified amount of H2O2. Good luck with your experiments.
Tablets for 5 ml:
http://www.sigmaaldrich.com/catalog/product/sigma/d4293?lang=en®ion=GB
Hallo Elena, as Maria already mentioned your concentration is to high. I prefer to prepare the DAB solution freshly because of the oxidation of air oxigen. Here is the protocol: solve 5 mg DAB in 10 ml PBS or TBS (ca. 10 min), filter the solution. Use a 10 ml syringe with Luer/Lock conus and a syringe filter (pore size 0,2 µm). Add 100 µl of a prediluted H2O2-solution (0,33/10ml 30%H2O2. Mix it well and react your sections either on the slides or free floating 10 Min up to 20 min. The filtered solution should be water clear. If the solution shows any red or brownish color or clouds don't use it. May be you can prepare a bigger amount , aliquotate and deep freeze it an -20°C. and it works, but I have no experience with this. Good luck, Ute
Thanks for the reply everyone! Yes I'm preparing a very high concentration of DAB but according to the datasheet, the DAB is soluble in water at 50mg/ml, and should produce clear to slightly hazy solution.
I on the other hand was using 1g in 40ml (half of the concentration). Anyway I tried staining with the clear/yellow DAB filtrate that I prepared last week and I did see some staining!. BTW this is how I prepare the DAB solution from the aliquoted concentrated DAB that I prepared :
5 ml................0.1M PB
200ul..............3.5% NiCl
1.5ml.............H2O2
500ul.............Concentrated DAB
I was getting too much background, for my next optimization I will be using less concentrated DAB.
You can disolve 100mg of DAB (one small bottle contains this amount of powder, Wako Chemical Co., made in Japan) by adding 4ml water and wait for about one hour. The solution is clear and no color. The solution can be kept in -20C for a long time. I have been doing this procedure for more than 30 years and no problem. The manufacturer is important. Do not measure the amount of DAB powder, because toxic. After the experiment, you need to add a bleech to the solution for detoxification.
Dear Elena, Nobuteru mentioned a very important issue - it is crucial to use chromogens from a source you know that produces good quality reagents. Another important point - filtering. When I use DAB tablets (or any other chomogen) I always filter the solution through a syringe filter (pore size 0.045 micrometre), even it looks clear to the naked eye. As you add nickel chloride as DAB enhancement, I would add filtering as the last step.
Hi Maria and Nobuteru, we purchased the DAB from Sigma. However, I was not consulted when the purchase was done, hence, the powder was ordered instead of a kit (which I would prefer).
BTW Maria, you would filter the DAB mixture again after the addition of Nickel Chloride? Maybe I can try that, because I just visualized the slide that I incubate for 2 min with :
0.1M PB................5 ml
30% H2O2.............0.75ul
25ug/ml DAB.........100 ul
3.5% NiCl..............100 ul
A lot of background staining observed. Will try filtering after mixing everything tomorrow.
Hi Elena, if you like to use an enhancement with Ni I recommand to use TBS. Here my protocol:
0,05 M TRIS pH 8...................................40 ml
DAB..........................................................8 mg
Ni-Ammoniumsulfatr...........................160 mg
solve and filter
Use 20 ml for 5 min preincubation
ad 10µl H2O2 (30%) to 20 ml DAB; Inkubation time: 10 min or longer; check it under the microscope.
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