H&E (hematoxylin and eosin) stains

[Claisen]

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 :)

 

[Monique]

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.

 

[Claisen]

Hi Monique, thanks for your reply :)

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

Anyone got any ideas?
Claisen

 

[chemisttree]

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.

 

[Claisen]

Hi Chemisttree,

Thanks for help!. Isn't NaIO₃ usually added (when the soln is initially prepared) to oxidize the Haematoxylin?

 

[chemisttree]

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.

 

[Claisen]

Ah ok. Thanks a lot for your help

 

[chemisttree]

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.