osmiumtetroxide enhancement...CORRECTION

[sarah.pixley at uc.edu]

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 
>>present
>>>>>but weak.  I've read that one can use osmium tetooxide to enhance 
>>visualisation
>>>>>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 
>>out,
>>>>>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.

>>Andrew Ray
>>Emory University
>>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.

>Good luck!

>S. Pixley
>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.

Apologies!!
S. Pixley