H&E (hematoxylin and eosin) stains

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Discussion Overview

The discussion revolves around the molecular mechanisms involved in hematoxylin and eosin (H&E) staining, particularly focusing on the "blueing" process and the chemical interactions at play. Participants explore the roles of various compounds, the effects of pH on color changes, and the oxidation states of the staining agents.

Discussion Character

  • Exploratory
  • Technical explanation
  • Debate/contested

Main Points Raised

  • One participant suggests that Haematein complexes with Al3+ and binds to negative residues like the phosphate backbone of DNA, raising questions about the molecular changes during "blueing".
  • Another participant notes that the aluminum complex is sensitive to pH changes, resulting in a purple color in acidic conditions and a blue color in alkaline conditions, but defers to chemists for further explanation.
  • A different participant mentions that their haematoxylin solution appeared red, prompting speculation about the color change upon binding to chromatin.
  • One participant proposes that the reduced form of hematoxylin is red while the oxidized form, Hematein, is blue, linking this to the common oxidation of phenols to quinones under basic conditions.
  • There is a question about the role of NaIO3 in the staining process, with one participant suggesting it is used to oxidize Haematoxylin, while another later contradicts this, indicating it is meant to oxidize hematein contaminants.

Areas of Agreement / Disagreement

Participants express differing views on the role of NaIO3 and the oxidation process of Haematoxylin, indicating that there is no consensus on this aspect of the discussion. Additionally, there are varying interpretations of the color changes associated with the staining process.

Contextual Notes

Some assumptions regarding the chemical interactions and the specific roles of compounds like NaIO3 remain unresolved. The discussion also highlights the dependence on pH and oxidation states, which may not be fully clarified.

Claisen
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Anyone know exactly what is happening at the molecular level during a H&E stain - particularly during "blueing"?
AFAIK (and I may be wrong), Haematein is complexed with Al3+ (assuming the mordant is alum), and that complex binds to -ve residues like the phosphate backbone of DNA. During "Blueing", ammonia solution (or some alkaline soln) is added, and the stain becomes blue. Anyone know why? What happens?

Hope someone can help :)
 
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The aluminum gives the hematoxylin a purple-to-blue color, the complex is sensitive to pH-changes, it has a purple color in acidic solution and a blue color in alkaline solution. You'd have to ask a chemist where this pH-sensitivity comes from, so I'll move this topic to that subforum.

p.s. The reason you want to have the color blue and not purple is because you want maximal contrast with the counterstain, which is usually red.
 
Hi Monique, thanks for your reply :)

I did a H&E stain last week, and the (Ehrlich's) haematoxylin solution actually looked red! :bugeye: I didn't check under the microscope, perhaps the complex is purple once bound to chromatin?

Anyone got any ideas?
Claisen
 
It looks to me like the reduced form of the stain (hematoxylin) is red and the oxidized form of the stain (Hematein) is blue. You will note that the structure of Hematein (blue) is a quinone. It is very common for phenols to be easily air oxidized to darkly colored quinones under basic conditions.

hematoxylin - http://en.wikipedia.org/wiki/Hematoxylin
hematein - http://en.wikipedia.org/wiki/Haematein

Note the rightmost ring of the system as shown in Wikipedia.
 
Hi Chemisttree,

Thanks for help!. Isn't NaIO3 usually added (when the soln is initially prepared) to oxidize the Haematoxylin?
 
I don't know if NaIO3 is added or not but if it is, it is done so to oxidize any hematein contaminant that might be present in the haematoxylin reagent, not to oxidize the haematoxylin.
 
Ah ok. Thanks a lot for your help :smile:
 
chemisttree said:
I don't know if NaIO3 is added or not but if it is, it is done so to oxidize any hematein contaminant that might be present in the haematoxylin reagent, not to oxidize the haematoxylin.

Now that just doesn't make any sense at all! Forget that I wrote that ... you had the right idea. The NaIO3 is added to oxidize the haematoxylin.