FW: [Histonet] Evans Blue and immunhistochemistry

From:"Monson, Frederick "

Morning Katrin,
	Evans Blue is known for its stochiometric reaction with albuminoids.  Thus, it can be used for tests in the following manner.
	1.  One can inject the dye, in which case it binds with protein and nicely demonstrates extravasation of blood, etc.
	2.  One can go to the trouble of purchasing rabbit albumin or isolating it from rabbit blood (3.5+g/100ml) and reacting the Evans Blue in vitro before reintroducing it as an indicator.

	Because of its binding characteristics, other than a bleach (I have no knowledge of any such use), there is little hope to remove the dye.
	I prior work with rabbits and extravasation, we have also used Indigocarmine which does not bind to the tissues and readily leaches out of the tissue.  
	Other help

Why Certain Dyes Are Useful for Localizing the Sentinel Lymph Node 
Tsopelas and Sutton J Nucl Med.2002; 43: 1377-1382. http://jnm.snmjournals.org/cgi/reprint/44/9/1540.pdf

Evans Blue is included in some Ab kits as a counterstain, thus, it would appear that its presence would not affect Ab binding.  This, however, should be tested by you to demonstrate that EB has no effect on Ab reactions.  

Rasio, E., Hampers, C. L., Soeldner, J. S. and Cahill, G. F (1967). Diffusion of glucose, insulin, inulin and Evans-blue protein into thoracic duct lymph of man. J. Clin. Invest 46, 903-910.

I searched Google with  to find the above.

Hope this helps and Merry Christmas,

Frederick C. Monson, PhD
Center for Advanced Scientific Imaging
Mail to Geology
West Chester University of Pennsylvania
Schmucker II Science Center, Room SS024
South Church Street and Rosedale Avenue
West Chester, PA, 19383
Phone/FAX:  610-738-0437
eMail:  fmonson@wcupa.edu
CASI Page and Scheduling

-----Original Message-----
From: Foerster, Katharina [mailto:foerster@CH11.UKL.UNI-FREIBURG.DE] 
Sent: Tuesday, December 16, 2003 6:25 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Evans Blue and immunhistochemistry

Dear Histonetters,

I`m looking for information about the parallel use of Evans Blue (to 
identify the area at risk induced by coronary artery ligation) and 
TTC (to identify the infarct zone) with a following immuno staining 
for vessels (anti-smooth-muscle-actin-antibody and cd31). Additionally, I wish to use Haematoxylin-Eosin staining and 
Elastica-van-Gieson in the heart-tissue.

Evans Blue and TTC are necessary for macroscopic infarct size 
assessment and not replacable in this project.
But it will be possible to take the tissue samples before TTC 
staining, so only Evans Blue (4 - 5 ml 10% dye per vena jugularis 
of the rabbit) remains problematical. 

I found some information about Evans Blue and H & E staining and 
this combination seems to be possible.

But I`m not sure if Evans Blue interferes with or covers any of the 
other stainings. 
If so, is there a possibility to wash out Evans Blue before 
immunhistochemistry (heart tissue will be fixed in 4% 
formaldehyde before embedded in paraffin)? Or is the microscopic 
tissue discolouration negligible because of albumin-binding?

Thank you for your help.

Katharina Förster
University Hospital Freiburg
Dept. Heart and vessel surgery
79106 Freiburg
e-mail: foerster@ch11.ukl.uni-freiburg.de

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