AW: [Histonet] bluing hematoxylin and alkaline water????

From:"Gudrun Lang"



Is this a Haematoxylin-Eosin-stain? If so, I think you wash out the Eosin
during the long water-wash. That makes the difference.

Haemalaun binds to negative charged tissueparts best in acid solution
(pH3-4). With this pH the colour is redish. Blueing means to elevate the pH
about 7-8. Now the bound dye-molecule is remodeld. The colour turns blue and
is more stable.
Gudrun

-----Ursprüngliche Nachricht-----
Von: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] Im Auftrag von Eva
Permaul
Gesendet: Freitag, 21. November 2008 17:11
An: histonet@lists.utsouthwestern.edu
Betreff: [Histonet] bluing hematoxylin and alkaline water????

Good morning out in histoland,
Thank god it is Friday. Have just a small question that I am sure you 
all have an answer for.
It is about bluing slides after hematoxylin. I read somewhere that 
alkaline water can blue hematoxylin and I am wondering if this is part 
of our problem. We use an automated stainer that we run both during the 
day and during the night. Normally we blue the slides in 1% ammonium 
after the runs for 1min. The run that was done during the day ended up 
less blue (more purple). The ones that run during the night continue 
being washed in water until we come in in the morning. They ended up 
more blue. I tested the pH of the water we are using. It is 7.96. Would 
this be enough to blue the slides if they are washed in it every hour 
from 9pm to 8am? Any other reason this might be happening? Should we not 
blue the slides that are run during the night? Why do we blue 
hematoxylin anyways?
Thank you for all your answers
Eva Permaul
(still learning)

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