Re: immunostainig

<< Previous Message | Next Message >>
From:Connie McManus <conmac@cc.usu.edu>
To:Garry Ashton <GAshton@picr.man.ac.uk>
Reply-To:
Content-Type:multipart/alternative; boundary=------------0981D23E7BB7DDD815C6ADC4


--------------0981D23E7BB7DDD815C6ADC4
Content-Type: text/plain; charset=us-ascii
Content-Transfer-Encoding: 7bit

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

--------------0981D23E7BB7DDD815C6ADC4
Content-Type: text/html; charset=us-ascii
Content-Transfer-Encoding: 7bit

<!doctype html public "-//w3c//dtd html 4.0 transitional//en">
<html>
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>
</html>

--------------0981D23E7BB7DDD815C6ADC4--




<< Previous Message | Next Message >>