RE: blue mucin in H&E

From:Gayle Callis <>

Jerry Fredenburgh, Richard Allan Scientific, also lectures that aluminum
sulfate mordant in the hematoxylin permits mucin staining compared to other
mordants, potassium aluminum sulfate.  Mordants do acidify hematoxylin, but
acetic acid is commonly added to bring the pH range into approx 2.3- 2.9.

Bert give some excellent advice here about regressive versus progressive,
with regressive methods being more difficult to control.  Harris originally
used his hematoxylin as a progressive stain, and didn't try regressive
staining until later.  YOU can do Harris as progressive, but timing in
stains, etc are reduced.  Surgipath Harris hematoxylin comes with two sets
of directions, one regressive as we have all learned to do it, and the
other progressive. 

An excellent publication discussing the mechanisms of hemalum staining and
Harris hematoxylin by Meloan and Puchtler is found in Journal of
Histotechnology, Dec 1987.  An eye opener on how hemalum stains work.
Understanding the effects of acid, pH (Elias, American J of Medical
Technology 40:513-514, 1974), etc are very important, plus some good

Also, if progressive hematoxylins are being used, acid alcohol
differentiation step is not needed (that is the technic FOR REGRESSIVE
Harris hematoxylin).  Gill talks about this in his publication and most
commercial reagents say to use the acetic acid as a clarifier, to remove
background staining. One can also simply stain and rinse well, with proper
timing for most progressive hematoxylins, certainly Mayers is a good example.

Histonet archives also has commentary from Gary Gill and others about
hematoxylin, pH, etc some time ago, worth a look.




At 09:02 AM 10/5/00 -0400, you wrote:
>We have adjusted several protocols over the years for this type of 
>automation. Most recently we dispensed with a regressive technique and were 
>able to improve consistency and shorten the staining protocol by about 15 
>Two suggestions: If you wish to stay with a regressive technique, try 
>substituting aqueous acetic acid for acid alcohol. You may have to play 
>with the concentration a bit but consistency is generally improved because 
>small changes in the acetic concentration do not have the same impact as 
>small changes in acid alcohol concentration--basically its more forgiving. 
>When you are working with very weak acid alcohol it is difficult to 
>maintain the same exact concentration from day to day.
>Second, the mucin staining is a function of the Hematoxylin pH. Establish a 
>rigorous QC for Hematoxylin before use. Lowering the pH (usually by adding 
>acetic acid) can significantly reduce mucin staining. Automation of this 
>type can really exaggerate even small differences in the Hematoxylin. 
>noticeable differences occur with pH changes of as small as 0.2 either 
>Bert Dotson
>-----Original Message-----
>From:	a i d a n   s c h u r r []
>Sent:	Wednesday, October 04, 2000 9:52 PM
>Subject:	blue mucin in H&E
>Hi all,
>We have recently switched to a linear staining system, which
>requires set, identical time in each solution.  To facilitate this, the
>concentration of the acid alcohol was lowered from 1% to 0.2%.
>We are now experiencing blue staining of mucin in cervical, colonic
>and gastric biopsies.  Any thoughts?!
>Aidan, New Zealand
>shin: device for finding furniture in the dark...
>a i d a n   c   s c h u r r
>     section head,  histology department
>      hutt valley health
>       high street
>        lower hutt, new zealand
>     ph.  ++64 4 5709173
>     fax  ++64 4 5709214
Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303

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