osmiumtetroxide enhancement of immunocytochem

sarah.pixley at uc.edu sarah.pixley at uc.edu
Sun Aug 20 15:00:24 EST 1995


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  




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