Re: immunostainig
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From: | Connie McManus <conmac@cc.usu.edu> |
To: | Garry Ashton <GAshton@picr.man.ac.uk> |
Reply-To: | |
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You may want to try a different hematoxylin formulation. I get very pale to
nonexistent hematoxylin counterstaining on the nuclei of the transgenic mice I
do HBV IHC on.. I was using Mayer's Acid Hemalum which worked great on other
tissues, but not on these transgenic mouse livers. I did some experimenting and
came up with my own version of a non alcoholic Harris' Hematoxylin and it works
fabulously. I found that it needs to ripen for about 1 week for the best
results. I haven't decided if it actually ripens or if the hemtoxylin crystals
dissolve completely by that time. Anyway, it's easy to do: prepare Harris
hemtoxylin omitting the alcohol. Using a mortar a pestal, grind the hemtoxylin
with a very small portion of the DI water used in the forumla, then add to the
alum solution and boil for 1 1/2 minutes rather than 30 seconds.. Filter before
each use. I counterstain for 3 minutes, rinse, then place in either ammonia
water or a saturated lithium carbonate solution to blue. If they are too
overstained, I let them soak in DI water until the nuclei are distinct (watch
with a microscope). Hope this helps you.
Connie McManus
Garry Ashton wrote:
> Dear all,
> Has anybody out there had any experience of a haematoxylin counterstain
> being prevented from working, after immunostaining with an antibody. The
> researcher presenting the problem doesn't know whether the Ab is nuclear or
> cytoplasmic marker.
> It cannot be fixation or necrotic tissue causing the problem as the negative
> controls apparently stain fine as does her staining with other antibodies.
> Any suggestions are welcome.
> Garry
>
> Paterson Institute
> Wilmslow Road
> Manchester
> M20 9BX
> UK
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You may want to try a different hematoxylin formulation. I
get very pale to nonexistent hematoxylin counterstaining on the nuclei
of the transgenic mice I do HBV IHC on.. I was using Mayer's Acid
Hemalum which worked great on other tissues, but not on these transgenic
mouse livers. I did some experimenting and came up with my own version
of a non alcoholic Harris' Hematoxylin and it works fabulously.
I found that it needs to ripen for about 1 week for the best results.
I haven't decided if it actually ripens or if the hemtoxylin crystals dissolve
completely by that time. Anyway, it's easy to do: prepare Harris
hemtoxylin omitting the alcohol. Using a mortar a pestal, grind
the hemtoxylin with a <u>very small</u> portion of the DI water used in
the forumla, then add to the alum solution and boil for 1 1/2 minutes rather
than 30 seconds.. Filter before each use. I counterstain for 3 minutes,
rinse, then place in either ammonia water or a saturated lithium carbonate
solution to blue. If they are too overstained, I let them soak in
DI water until the nuclei are distinct (watch with a microscope).
Hope this helps you.
<p>Connie McManus
<p>Garry Ashton wrote:
<blockquote TYPE=CITE>Dear all,
<br>Has anybody out there had any experience of a haematoxylin counterstain
<br>being prevented from working, after immunostaining with an antibody.
The
<br>researcher presenting the problem doesn't know whether the Ab is nuclear
or
<br>cytoplasmic marker.
<br>It cannot be fixation or necrotic tissue causing the problem as the
negative
<br>controls apparently stain fine as does her staining with other antibodies.
<br>Any suggestions are welcome.
<br>Garry
<p>Paterson Institute
<br>Wilmslow Road
<br>Manchester
<br>M20 9BX
<br>UK</blockquote>
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