Immunostaining of mouse heart

Dr Engelbert Buxbaum via methods%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Thu Jan 24 16:15:46 EST 2008


Am 21.01.2008, 17:03 Uhr, schrieb ana <ana-irda_ from hotmail.com>:

> If i want to stain a slide with heart tissue of a mouse what method  
> should I use?
> I want to clearly see the small coronary vessels.

Just HE or any of the trichromes should do, but you will have to search  
under the microscope for the vessels. Better would be to replace the blood  
in the vessels by stained gelatine:

Put an animal under terminal narcosis with Phenobarbital. When it no  
longer reacts to pinching of the foot open the chest, cut a small hole  
into the bottom of the left ventricle with a scissor and insert a blunt  
needle through this hole up into the aorta. Clamp it there with a  
hemostat. Cut open the Vena cava, so that fluid can drain. Then infuse PBS  
through the needle (possibly also glutardialdehyde solution to fix the  
tissue) and later the stained, warm gelatin solution. Once the stained  
solution comes out at the vena cava stop the perfusion. Cut out the heart  
(and any other tissue you may need) and (post-)fix, embed and cut in the  
usual way. Use a counterstain that contrasts well with the stained gelatin.

When you do the perfusion do not be startled by the cramping movement you  
see in the animal, this comes from changes in the ion environment of the  
muscle and has nothing to do with brain activity.

I have done similar things with rats with an 18G needle, for mice of  
course you need something smaller. Cut away the sharp tip and grind the  
end smooth, so that you don't puncture the aorta, since then you would  
loose the animal.

Instead of gelatin you may also use the monomer of a suitable plastic.  
Once the plastic has hardened you can mazerate away the tissue and you are  
left with the blood vessels only: that looks absolutely amazing. No  
embeding and cutting required.




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