Eva:
Hematoxylin is a pH indicator, it turns reddish when you differentiate it in the acid alcohol, and returns to "violet-blue" when you either "blue" it with the ammonia water or place it in tap water after the alcohol-acid solution.
The differentiation you know is to make the nuclear details more visible and the bluing is essentially done to stop the acid effect of the clearing, that could end decolorizing the section.
It is a good practice to "blue" with the alkaline (ammonia) solution or just plain tap water if it is "hard" enough (containing salts), but it is not good practice to let the slides in running water for long periods of time because, being the "universal solvent", running water will end making the stain fainter, as you have noticed in the difference between the two types of batches you mention.
It would preferable to leave the last batch in your clearing (pre-coverslipping) liquid (usually xylene), this will not affect the staining intensity at all.
René J.
--- On Fri, 11/21/08, Eva Permaul wrote:
From: Eva Permaul
Subject: [Histonet] bluing hematoxylin and alkaline water????
To: histonet@lists.utsouthwestern.edu
Date: Friday, November 21, 2008, 11:11 AM
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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