[Histonet] Stain recipe

[idimitro <@t> mun.ca]

Hi Catherine, 

I have staining protocol for alcian blue and nuclear fast red, see bellow. I looked in Conn's biological stains book (9th edition,p.188)and saw that chlorantine
fast red 5B is soluble in water. I think you can substitute Nuclear fast red with chlorantine
fast red 5B and try and see the results. Connective tissue will be deeper red. Use controls and try it.

Iliana Dimitrova, MSc, RT
Histology Supervisor
Medical Education and Laboratory Support Services (MELSS)
Faculty of Medicine
Memorial University of Newfoundland
St. John's, NL Canada 

Alcian Blue Staining Protocol
 
NovaUltra Special Stain Kits
 
Description: Alcian blue stains acid mucosubstances and acetic mucins. Excessive amounts of non-sulfated acidic mucosubstances are seen in mesotheliomas, certain amounts occur normally in blood vessel walls but increase in early lesions of atherosclerosis. Strongly acidic mucosubstances will be stained blue, nuclei will be stained pink to red, and cytoplasm will be stained pale pink. 
  
Fixation: formalin fixed, paraffin embedded tissue sections. 
  
Solutions and Reagents: 
  
3% Acetic Acid Solution: 

      Glacial acetic acid ----------------- 3 ml 
      Distilled water --------------------- 97 ml 
  

Alcian Blue Solution (pH 2.5): 

      Alcian blue, 8GX -------------------- 1 g 
      Acetic acid, 3% solution ----------- 100 ml 
      Mix well and adjust pH to 2.5 using acetic acid. 
  

0.1% Nuclear Fast Red Solution: 

      Nuclear fast red ------------------- 0.1 g
      Aluminum sulfate------------------ 5 g 
      Distilled water ---------------------100 ml 
      Dissolve aluminum sulfate in water. Add nuclear fast red and slowly heat to boil and cool. 

Filter and add a grain of thymol as a preservative.


Procedure: 
  
1.       Deparaffinize slides and hydrate to distilled water. 
2.       Stain in alcian blue solution for 30 minutes. 
3.       Wash in running tap water for 2 minutes. 
4.       Rinse in distilled water. 
5.       Counterstain in nuclear fast red solution for 5 minutes. 
6.       Wash in running tap water for 1 minute. 
7.       Dehydrate and through 95% alcohol, 2 changes of absolute alcohol, 3 minutes each. 
8.       Clear in xylene or xylene substitute. 
9.       Mount with resinous mounting medium. 
  
Results: 
  
      Strongly acidic sulfated mucosubstances -------------- blue 
      Nuclei ------------------------------------------------------ pink to red 
      Cytoplasm ------------------------------------------------- pale pink   
 
      
Positive Controls: 
  
      Small intestine, appendix, or colon.

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Ross, Catherine
Sent: December 13, 2011 5:45 AM
To: histonet <@t> lists.utsouthwestern.edu
Cc: Grainger, Shirley
Subject: [Histonet] Stain recipe

All,
 
Greetings from across the pond!
 
One of our histology technicians is looking for a recipe for an alcian
blue/sirius red stain, otherwise known as "Alcian blue 8G with chlorantine
fast red 5B". It is not one I have used but I suggested tapping into the
wealth of knowledge on this forum - can anyone help, please?
 
Thank you in advance,
Catherine
 
 
Dr. Catherine L. Ross  DVM MSc(VetPath) MRCVS
Covance Laboratories (UK) Ltd.
Otley Road,
Harrogate, N. Yorks.
HG3 1PY
 
T: +44 (0) 1423 848759
E: catherine.ross <@t> covance.com <mailto:catherine.ross <@t> covance.com> 
 


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