[Histonet] SMC Immuno and Elastin combo?

[Tony Henwood (SCHN)]

Nancy,

The red of the classic VG counterstain stains the collagen. Muscle will appear yellow. If the yellow is obscuring the brown DAB then after VG staining wash the slides in water. This will remove the picric acid staining leaving the muscle brown.
Since Nova red is red (I assume since I have not had cause to use it - I'm a DAB man!), I would use Picro Indigo carmine or Picro-Light green.

Picro-indigo Solution (Ortiz-Hidalgo C "Abelardo Gallego(1879-1930)and his contributions to histotechnology: The Gallego stains" Acta histochemica1 13(2011):189-193):
Picric acid (saturated solution)	100ml
Indigo carmine			0.25mg
Distilled water			15ml

Whippy's Elastic Stain (Whippy P "A Stain to Try - Whippy's Elastic Stain" Histograph Jan 2008:7):
Light Green Stock Solution                                                              
  Light Green  		2 gm                 
  Distilled water  	  	100 ml                   
  Glacial Acetic Acid  	  2 ml                  
  Place Light Green and distilled water into a conical flask and mix well.                                                                         
  Add Acetic acid and mix.                                                              
  Filter and pour into stock bottle.

Light Green - Picrate (Whippy's) Solution 
  Saturated aqueous picric acid  	  	10 ml                 
  Light Green stock solution		5 drops                                       
  Pour Picric Acid into a conical flask.                                               
  Add Light Green solution, stir until dissolved.

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Laboratory Manager & Senior Scientist 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Thomas, Nancy
Sent: Thursday, 14 March 2013 3:33 AM
To: 'Patricia Bourassa'; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] SMC Immuno and Elastin combo?

Patti,
The immunostaining probably was not wiped out, rather just covered up with the red color of the VG counterstain.  You might try a chromogen that expresses in a different color.  Or, you might leave off the van gieson part of the EVG (staining only the elastic tissue) and pick a counterstain of another color.  Basically, you just need to make sure that none of the stains used have the same color outcome.  I have done this combo in the past.  Never really finished the project because the issue I had was that the lizard tissue I was working with just would not stay on the slide no matter what I tried.  But, I do remember that I treated them as two separate stains.   I did the actin staining first, rinsed it well, then started in on the elastin stain.  You probably have a protocol for each of these and you just need to combine them. 

Nancy Thomas	
Stowers Institute

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Patricia Bourassa
Sent: Wednesday, March 13, 2013 10:58 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] SMC Immuno and Elastin combo?

Hello,

I've read some papers which reference the combination of smooth muscle cell actin immunostaining with a Verhoeff's elastin counterstain.

Has anyone ever done this and if so, do you have protocol you'd be willing to share?

I'm staining mouse and rat lung and have a SMC protocol working that uses
an Abcam antibody and NovaRed chromagen (peroxidase).   I've also done EVG
staining so I'm familiar with the general process.  The combination is what I'm not sure of - I gave it a try for the heck of it and wiped out the immunostain.

We've just started looking at this, so nothing is set in stone and I'd be grateful for any advice/ procedural help.

Thanks in advance!

Patti Bourassa
Karos Pharmaceuticals
--
*Patti Bourassa
Senior Scientist
Karos Pharmaceuticals
(203) 535-0540, ext 207*
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