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Author: Subject: H&E Stain
exodia
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[*] posted on 7-10-2016 at 10:17
H&E Stain


Hello there, I´m starting on microscopy (rather proper microscopy, I have been playing around with the microscope ever since I was a kid but now I´m more interested in stains and embedding techniques)

I have acquired some hematoxylin and eosin powder and I am planning on preparing the stains myself, i just wanted to ask if any of you have any experiences preparing them from scratch.

eosin Y (from cshprotocols.org):

1. Prepare eosin Y stock solution. Add 2.0 g of water-soluble eosin Y to 40 mL of double-distilled H2O, and mix until dissolved. Then add 160 mL of 95% ethanol, and mix. Store at room temperature.

2. Prepare eosin Y working solution. Add 200 mL of eosin Y stock solution to 600 mL of 80% ethanol and mix well. While working in a fume hood, add 4 mL of glacial acetic acid and mix well. Store covered at room temperature.

surprisingly I couldnt find lots of info regarding how to prepare it properly, anyone knows if the working solution degrades over time? or there is another reason to have a stock, and then converting it to the working solution? (also I know it´s a strong dye but 2g in about 800ml of final volume seems rather weak.

hematoxylin (from nottingham.ac.uk):

Haematoxylin 10g
Absolute Alcohol 100ml
Potassium Alum 200g
Distilled Water 2000ml
Mercuric Oxide 5g

Dissolve the haematoxylin in absolute alcohol in a small beaker. Dissolve the potassium alum in warm distilled water in 5L flask and bring to the boil. Remove from the heat and add the mercuric oxide and cool rapidly in a sink full of iced water. Place into a dark glass winchester.

Before use, add 0.5ml acetic acid to 60ml haematoxylin solution, then filter into a staining dish. The remainder of the solution is stored in a cool place until needed.

the only problem I see with the hematoxylin its mercuric oxide, really?? (I have looked around and most of the recipes I found seem to have mercuric oxide, one of them even has mercuric chloride wich is even more unappealing.

I did find another variant called the "mayers hematoxylin" wich uses more or less the same compunds and sodium iodate un sustitution for de mercury.

Is there a nicer way for long term storage without Hg? (wich surprises me because I remember doing the stain in school and no safety regarding Hg was mentioned) and it wasn´t that long ago.

Thank you.

[Edited on 7-10-2016 by exodia]
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Metacelsus
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[*] posted on 7-10-2016 at 11:44


The mercuric oxide is used as an oxidizing agent to convert the haematolyxin into the active form (hematein). You could probably substitute another mild oxidizer; see here: http://www.leicabiosystems.com/pathologyleaders/the-basic-ch...



As below, so above.
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exodia
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[*] posted on 7-10-2016 at 14:03


thank you metacelsus, I did see they recomend as well the sodium iodate I´ll probably go with that one to try and stay mercury free.
any idea for how long they can be stored before degradation?

I´ll have some stock, but when I do activate it, it seems like a waste just staining a set of slides and throwing away the solution.
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