[Histonet] H&E+ Alcian Blue

[Tony Henwood]

Debbie,

It seems that the eosin is masking the alcian blue staining (ie changing
the colour of the alcian blue). Try differentiating (or removing) the
eosin by prolonged washing in water after the eosin stain. Beware this
might occur quite quickly so monitor it microscopically. If you
inadverrtantly over-differentiate, then just restain in the eosin and
differentiate again. The eosin counterstain should not be too strong
(not as vivid as your normal H&E).

It is also advisable to choose a puple Hx (like Harrises) rather than a
blue HX (like Mayers). I am not sure what Haematoxylin Z is.

Regards

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead 
Cnr Hawkesbury Road and Hainsworth Street, Westmead 
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 




-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Deborah
Faichney
Sent: Thursday, 9 September 2010 6:45 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] H&E+ Alcian Blue


Hello all,

I have a request to carry out a combined staining with H&E + Alcian Blue
pH2.5.    I have tried in vain to get this to work but regardless of the
order of staining the end result is dark blue/purple mucin.  I have
carried out a parallel experiment whereby the staining has been checked
microscopically then stopped after each of the dyes.   (Thus giving 3
slides stained with:  AB, AB+H and AB+H+E)  The AB and AB+H are really
nicely stained but as soon as the eosin is added (using 2 different
stains and a variety of times) the mucin staining looks similar to the
nuclear stain.  I am expecting the alcian blue to remain turquoise:
should it?

For information, I am trying to stain salmon intestine at 5 microns for
using the following:

Alcian Blue 8GX (certified), pH has been checked
Haematoxylin Z (Cellpath Uk)
1% aq Eosin (Cellpath uk) and lab prepared solution from dye.

Thanks from a frustrated technician!

Debbie Faichney
Histopathology
Institute of Aquaculture
University of Stirling
Scotland
UK








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