RE: H&E staining

From:Evelyn Kaplan

Hi Kevin,
First of all, what exactly are you trying to do? Is this for routine H&E staining on paraffin processed tissues? By the looks of what you have written, and it it sounds very confusing, you should use only one type of haematoxylin at once. They each have different purposes and you are wasting valuable reagents trying to mix the properties/mechanisms of the two.
Secondly; I am unsure what "Clear Rite" is but if you are using regressive haematoxylin, then a differentiator of 0.5% or 1% acid alcohol (depending on how fresh your stain is) in one pot is usually sufficient to differentiate Harris' haematoxylin staining of, say, ten minutes.
Thirdly; I assume the ammonium hydroxide is the 'blueing' agent and I use Scott's Tap Water Substitute in one pot and find that sufficient to blue perfectly well. All of the solutions I have mentioned can be found in Bancroft and Gamble (2002), Churchill Livingstone.
Using your reagents maybe try:
regressive haematoxylin in as many pots as necessary for an overall time of 10 mins followed by wash, differentiator, wash, blueing agent, wash (all one pot each) and then your eosin. Of course you may then have to 'tweak' this to get the exact staining you want/prefer.
Fourthly; if you want good constructive replies in response to your pleas for help, put a subject in the subject line. Most days I ignore anything without a subject but today the students are finished, the sun is shinning and I am having a good day!
Good Luck!

Evelyn Kaplan,
Dept of Pathology,
College of Medicine and Health Sciences,
Sultan Qaboos University,


 From: kevin williams []
Sent: Sunday, June 29, 2003 1:09 AM

Dear All:
I have tried out a system of a(first three) regressive Gills  Heaematoxylin, followed  by a Progressive Haematoxylin, of the same strenght and the slides are exposed to it for the same amont of time. The regressive is constantly dilueted by water and alcohol and the regressive is constantly exposted to the progressive Haematoxylin, due to the fact that I use a lini stainer.
Can you give me any insits into the good quality staining I have found using no differentiator(previously using Clear Rite) followed by Amonium Hydroxide. The eosin then follows, only gets 10 seconds. So the best I have found is without the Clear Rite and the Amonium Hydroxide. So please tell me what is going on and why. Why is most important.
Thanks for all your help,
Yours faithfully,
A. Kevin Willimas.

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