sarah.pixley at uc.edu
sarah.pixley at uc.edu
Sun Aug 20 17:02:27 EST 1995
sarah.pixley at uc.edu wrote:
>Andrew Ray <aray at emory.edu> wrote:
>>In article <40vpdo$m22 at agate.berkeley.edu>, <Noone at uclink.berkeley.edu> writes:
>>>>>I have done immuno against parvalbumin in brain tissue and staining is
>>>>>but weak. I've read that one can use osmium tetooxide to enhance
>>>>>of the precipitate from the DAB reaction. However, I need specific
>>>>>instructions about how to use the osmium to do this. If you can help me
>>>>>post in the
>>>>>news group or email me at kopl that is kopello at eden.rutgers.edu.
>>>>hi Michael, I can't provide the method but I thought you could do with
>>>>some OH & S stuff. Osmium tetroxide is mega- dangerous. It is both a strong
>>>>poison & mutagenic.
>>>I don't recommend using Osmium tetroxide to enhance DAB staining, unless you
>>want to investigate your tissue under the electron microscope.
>>We get excellent results with parvalbumin - fibers, cells, sometimes even
>>terminal fields. We use Ni ammonium sulfate, but don't have to to get good
>>staining. Are you sure you're using a high enough concentration of primary or
>>secondary (or tertiary if present) antibody? The other thing to wonder is if
>>you are using a long enough incubation in the DAB (or even in the Ab's). It
>>doesn't take much of a change in concentration or incubation to make a large
>>change in your labeling. Good Luck.
>>Dept of Neuroscience
>>aray at emory.ed
>To enhance DAB: 1) First try using a dark blue Whatten filter on your
>microscope, for photography, or dark field microscopy. Either will
>bring out details that your eye may not have detected in normal bright
>field microscopy. 2) We have found that the nickel/cobalt solution
>described below gives a significant boost to DAB staining. The
>reaction product is blue-black. While this can sometimes be a problem
>in thick tissues with cartilage, or where tissue edges and/or folds
>are present, it photographs nicely. Beware, this solution
>significantly shortens the reaction time. For example, if your normal
>DAB reaction time is one minute, then with the nickel/cobalt solution
>it could be around 15 seconds. After that time, the rest of the
>tissue turns a blue-grey and obscures your results. The stock
>solution and the DAB after adding the stock will have a precipitate.
>This does not seem to interfere with staining until it gets very
>heavy. Finally, the stock lasts a long time in the refrigerator, but
>it sometimes loses hydrogen peroxide. If it begins to be ineffective,
>add additional hydrogen peroxide or make a new batch.
>Nickel/cobalt for DAB enhancement:
> Into a final volume of 10 mls, using double distilled water, add:
> 300 ul of a 30% hydrogen peroxide solution
> 3 g NiCl
> 3 g CoCl
>Keep refrigerated. Add 1 drop (about 50 ul) to 10 mls of DAB (50
>mg/100 mls 0.1 M phosphate buffer).
>Of course, none of this will work if your primary antibodies or the
>other reagents are too dilute.
>University of Cincinnati
>sarah.pixley at uc.edu
I just tried to make this solution and realized that I made a mistake
in my message above!!!! The above ingredients should be added to a
volume of 100 mls, not 10 mls!!!!
SORRY ABOUT THAT!!!!
This formulation is from the Cell Proliferation Kit, catalog #RPN.20,
from Amersham. This solution is provided and all ingredients are
listed. We just proceeded to make it ourselves. The ingredients are
all at 3% weight or volume to volume (hydrogen peroxide, NiCl, CoCl).
Due reference should be given.
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