RE: Alcian Blue staining problem

From:"Smith, Allen"

The pH of your rinse water may vary with the pH of your water supply.
Alcian blue staining is much more uniform if the slide is rinsed in the solvent for the stain (0.1 N HCl or 3% acetic acid) rather than water.

Allen A. Smith, Ph.D.
Barry University
School of Graduate Medical Sciences
    Podiatric Medicine and Surgery
Miami Shores, Florida  33161-6695

-----Original Message-----
From: Manisha Deshpande []
Sent: Friday, October 04, 2002 1:05 AM
Subject: Alcian Blue staining problem

Dear HistoNet,
I am doing Alcian Blue staining according to the method given in the book "Manual of Histological Techniques and their diagnostic application" by John Bancroft & Harry Cook. I make Alcian Blue pH 1.0 by dissolving 1g Alcian Blue 8GX in 100 ml of 0.1N HCl and Alcian Blue pH 2.5 by dissolving 1g Alcian Blue 8GX in 100 ml of 3% acetic acid. I deparaffinize the slides in xylene and then hydrate them according to standard method. I then apply the stain for some time and then rinse with water.
The problem I am facing is that I am not getting reproducible results. One slide from a particular sample block gives blue staining while another slide from the same sample block does not get the blue color. Or one section on a slide gets blue color while the other two sections on the same slide do not get the blue color. My samples are uniform in nature, that is, if a particular sample is positive for Alcian Blue, it should be so throughout the sample, not in some parts. And if a sample is negative for Alcian Blue then it should be so throughout the sample, not in some regions. So every section cut from one sample block should give identical result.  
I would be grateful if I could get feedback on what might the problem be and what I should do to not get this variability in results. This has been tried independently by a colleague and the same thing is happening.
I would appreciate helpful clues in this regard. I do deparaffinization and hydration thouroughly so the problem does not appear to be incomplete deparaffinization of some sections. I have also tried the microwave technique but that also does not help. Other stains such as Eosin are not giving this problem with my samples - they are working fine.
I look forward to any suggestions.
Thanking you,
Dr. Manisha Deshpande,
Reliance Life Sciences,
Mumbai, India.

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