Discussion:
Suggestions for staining ground substances in the Heart
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Wolfe, Christina
2015-06-30 15:15:13 UTC
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Hi all,
We are interested in staining ground substances in the heart. Are there other stains that will work beside the Pentachrome? We have tried the Movat's pentachrome (commercial kit) and are able to demonstrate ground substance in the bone with the alcian blue part of this stain. In our hands the goblet cells of the gut and the ground substance in the heart are devoid of staining. We have tried sections cut at 4 and 6 microns. Any thoughts/suggestions?

Kristie

Christina Wolfe, BSHA, MLT (ASCP), HT, QIHC
Drug Safety Evaluation/Bristol-Myers Squibb
Pathology Dept.
812-307-2093


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Tony Henwood (SCHN)
2015-06-30 23:46:36 UTC
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If the ground substance and mucin are not staining, it would be a problem with the alcian blue solution.

Microscopically check the slides after the alcian blue step - if no or weak staining then:
1. Is the alcian blue at pH 2.5 or is it pH1 - (goblet cells in stomach and ground substance rarely stain at pH 1)?
2. Is the alcian blue dye of good quality. There are several out there - use Certified dyes.

Hopefully this helps

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Laboratory Manager & Senior Scientist, the Children's Hospital at Westmead
Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA


-----Original Message-----
From: Wolfe, Christina [mailto:***@bms.com]
Sent: Wednesday, 1 July 2015 1:15 AM
To: ***@lists.utsouthwestern.edu
Subject: [Histonet] Suggestions for staining ground substances in the Heart

Hi all,
We are interested in staining ground substances in the heart. Are there other stains that will work beside the Pentachrome? We have tried the Movat's pentachrome (commercial kit) and are able to demonstrate ground substance in the bone with the alcian blue part of this stain. In our hands the goblet cells of the gut and the ground substance in the heart are devoid of staining. We have tried sections cut at 4 and 6 microns. Any thoughts/suggestions?

Kristie

Christina Wolfe, BSHA, MLT (ASCP), HT, QIHC Drug Safety Evaluation/Bristol-Myers Squibb Pathology Dept.
812-307-2093


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John Kiernan
2015-07-02 05:49:40 UTC
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Movat's pentachrome is unnecessarily complicated when you want to look only at one component of a tissue.

Most "ground substance" (extracellular material between cells, collagen fibres etc) is weakly stainable with PAS, alcian blue pH2.5 or both. It's usual to stain with alcian blue first (Mowry's method). If you do the PAS first, the PAS-positive materials will also stain with alcian blue and be darker. For the reasons, see:

Johannes, M.-L. and Klessen, C. (1984). Alcian blue/PAS or PAS/alcian blue? Remarks on a classical technique used in carbohydrate histochemistry. Histochemistry 80:129-132.

Yamabayashi, S. (1987). Periodic acid-Schiff-alcian blue: a method for the differential staining of glycoproteins. Histochemical Journal 19:565-571.

Reid, P.E. and Owen, D.A. (1988). Some comments on the mechanism of the periodic acid-Schiff-Alcian blue method. Histochemical Journal 20:651-654.

If you can predict the PAS-AB effect without further reading, you already have a good understanding of how the traditional methods of carbohydrate histochemistry work!

John Kiernan
London, Canada
= = =
Post by Wolfe, Christina
Hi all,
We are interested in staining ground substances in the heart. Are there other stains that will work beside the Pentachrome? We have tried the Movat's pentachrome (commercial kit) and are able to demonstrate ground substance in the bone with the alcian blue part of this stain. In our hands the goblet cells of the gut and the ground substance in the heart are devoid of staining. We have tried sections cut at 4 and 6 microns. Any thoughts/suggestions?
Kristie
Christina Wolfe, BSHA, MLT (ASCP), HT, QIHC
Drug Safety Evaluation/Bristol-Myers Squibb
Pathology Dept.
812-307-2093
________________________________
This message (including any attachments) may contain confidential, proprietary, privileged and/or private information. The information is intended to be for the use of the individual or entity designated above. If you are not the intended recipient of this message, please notify the sender immediately, and delete the message and any attachments. Any disclosure, reproduction, distribution or other use of this message or any attachments by an individual or entity other than the intended recipient is prohibited.
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John Kiernan
2015-07-02 06:09:56 UTC
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Dear Histonetters,

I recently replied to a question about staining extracellular "ground substance" with alcian blue. Here is some more information.

With the alcian blue-PAS sequence it is necessary to have a good alcian blue dye - the same as or functionally equivalent to the original alcian blue 8G. For the PAS-alcian blue sequence, other alcian blues may also be OK.

Alcian blue from a batch certified (or rechecked) by the Biological Stain Commission (BSC) 5 or fewer years ago should be good for either procedure.

Of the dyes commonly used to make up staining solutions for microscopy, alcian blue is the only only one that sometimes deteriorates, unpredictably and often quite quickly, as a dry powder in the bottle. Some alcian blue powders from the 1950s are still OK, but some very much younger ones are no good 5+ years after passing the BSC's analytical and staining tests.

John Kiernan
London, Canada
http://biostain.com
= = =
Mayer,Toysha N
2015-07-02 14:57:04 UTC
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Kristie,
For heart you could try the elastic Trichrome, it will stain the ground substances and is faster than the Movat. If your ground substance is devoid of staining it could be the phosphotungstic acid. Is it 5%, and fresh? Are you using two changes at 5 min each? Continue with the 4micron sections, and use fresh solutions. If you can make the phosphotungstic yourself, do that. Commercial solutions sometimes do not store well.
Hope this helps.


Sincerely,

Toysha N. Mayer, D.H.Sc., MBA, HT (ASCP)
Instructor/Education Coordinator
Program in Histotechnology
School of Health Professions
UT M.D. Anderson Cancer Center
713.563-3481





-----Original Message-----
From: Wolfe, Christina [mailto:***@bms.com]
Sent: Wednesday, 1 July 2015 1:15 AM
To: ***@lists.utsouthwestern.edu
Subject: [Histonet] Suggestions for staining ground substances in the Heart

Hi all,
We are interested in staining ground substances in the heart. Are there other stains that will work beside the Pentachrome? We have tried the Movat's pentachrome (commercial kit) and are able to demonstrate ground substance in the bone with the alcian blue part of this stain. In our hands the goblet cells of the gut and the ground substance in the heart are devoid of staining. We have tried sections cut at 4 and 6 microns. Any thoughts/suggestions?

Kristie

Christina Wolfe, BSHA, MLT (ASCP), HT, QIHC Drug Safety Evaluation/Bristol-Myers Squibb Pathology Dept.
812-307-2093

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