[Histonet] Stain recipe. Nuclear fast red

Wed Dec 14 11:35:51 CST 2011

If the objects of interest are stained with alcian blue, any pink or red counterstain should be OK. Aluminium-nuclear fasr red if they want to see nuclei; eosin if a general pink to red background is sufficient. H&E is also OK after alcian blue because the two blue colours are clearly different. 

John Kiernan 
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On 14/12/11, idimitro <@t> mun.ca wrote:

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> John, 
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> No offence, there is so much in histology I don’t know, and any way I can learn is good. I use your textbook all the time.
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> In this case I meant to use chlorantine fast red 5B, which they want to use, instead of nuclear fast red, which is in my protocol.
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> What protocol would you suggest for them?
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> Thanks, 
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> iliana
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> From: John Kiernan [mailto:jkiernan <@t> uwo.ca] 
> Sent: December 14, 2011 2:31 AM
> To: Dimitrova, Iliana; Catherine.Ross <@t> covance.com; histonet <@t> lists.utsouthwestern.edu
> Cc: Shirley.Grainger <@t> covance.com
> Subject: RE: [Histonet] Stain recipe. Nuclear fast red
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> Sorry to make a blunt contradiction, because a few histonetters seem to take offence very easily. 
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> Nuclear fast red cannot replace any "chlorantine" dye.  The "chlorantine" dyes in the literature and lore of staining are anionic; they bind to proteins such as those in cytoplasm, collagen etc. 
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> The name nuclear fast red (Kernechtrot in German) goes with an anthraquinone dye long obsolete in the world of textile dyeing. It has a Colour Index (CI) number - 60760 - but it doesn't have a CI application name. Possibly the only use of this compound is as a biological stain.  It is used as its aluminium complex, which provides red staining of cell nuclei and weak background pink in cytoplasms and collagen. The Biological Stain Commission (BSC) certifies nuclear fast red.  
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> Vendors of kits that include aluminium-nuclear fast red as a nuclear counterstain should check that they are selling certified nuclear fast red.  The NFR standards were published in 2007.  
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> John Kiernan
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> Anatomy, UWO, London, Canada
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> (Secretary, Biological Stain Commission) 
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> On 13/12/11, idimitro <@t> mun.ca wrote: 
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> Hi Catherine, 
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> 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 
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> Alcian Blue Staining Protocol
>  
> NovaUltra Special Stain Kits
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> 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. 
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> Fixation: formalin fixed, paraffin embedded tissue sections. 
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> Solutions and Reagents: 
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> 3% Acetic Acid Solution: 
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>       Glacial acetic acid ----------------- 3 ml 
>       Distilled water --------------------- 97 ml 
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> Alcian Blue Solution (pH 2.5): 
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>       Alcian blue, 8GX -------------------- 1 g 
>       Acetic acid, 3% solution ----------- 100 ml 
>       Mix well and adjust pH to 2.5 using acetic acid. 
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> 0.1% Nuclear Fast Red Solution: 
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>       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. 
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> Filter and add a grain of thymol as a preservative.
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> Procedure: 
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> 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. 
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> Results: 
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>       Strongly acidic sulfated mucosubstances -------------- blue 
>       Nuclei ------------------------------------------------------ pink to red 
>       Cytoplasm ------------------------------------------------- pale pink   
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> Positive Controls: 
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>       Small intestine, appendix, or colon.
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> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] <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
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> All,
>  
> Greetings from across the pond!
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> 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?
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> Thank you in advance,
> Catherine
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> Dr. Catherine L. Ross  DVM MSc(VetPath) MRCVS
> Covance Laboratories (UK) Ltd.
> Otley Road,
> Harrogate, N. Yorks.
> HG3 1PY
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> T: +44 (0) 1423 848759
> E: catherine.ross <@t> covance.com <mailto:catherine.ross <@t> covance.com <catherine.ross <@t> covance.com>> 
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