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I've just learnt about staining during a histology class and I'm confused.

Why do we have to add acid alcohol and sodium bicarbonate to the tissue during Hematoxylin & Eosin staining? Is there any difference if we don't add them at all and just skipped to eosin instead?

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    $\begingroup$ Mind sharing the exact staining process that you are using? There are slight differences and honestly I have no idea what sodium bicarbonate would be for, unless it's an attempt to get some ions back into soft water used for the experiment, but study has shown that to be a very ineffective way to go about it. $\endgroup$
    – rotaredom
    Mar 6 at 14:34
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This is described with better detail (and better prose) in John Baker's classic description at StainsFile, but to summarize: The procedure you're describing is a regressive hematoxylin stain with Harris hematoxylin. To start off with, let's bear in mind that hematoxylin, a compound from the wood of the logwood tree, is actually used as hematein, by oxidizing it with sodium iodate. (The colorless hematoxylin would naturally "ripen", oxidize over 4-10 weeks in the presence of air, even without that step, but this is more controllable) Additionally, aluminum ions are added as a mordant, creating hemalum by binding =O and -OH on the hematein. The Al atom is what actually binds to something in the nucleus (I found reference to lysine side chains of histone proteins and the phosphate of the DNA - I haven't sorted this part out yet).

When acid is added, it competes with aluminum, which can be thought of as Al3+. The real bonding may be more covalent than that, but that point is considered controversial. For whatever reasons, the H+ interferes with the Al3+ mediated attachment of stain to tissue, especially at sites of background attachment (differentiation). The hematoxylin washes away somewhat, leaving lighter, more specific staining. (this tactic is called a regressive stain, and is certainly not needed to do the procedure. This is very much more art than science at this point...)

But ... the acidic hematoxylin has a reddish color, and it isn't as blue as it can be unless it is neutralized to around pH 8 - hence the second step with bicarbonate, though often plain tap water can be used. That is an ordinary instance of a dye changing color with pH, and can be called "bluing the hematoxylin".

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I'm just going to answer for the acid alcohol since I'm unaware of any purpose for using a sodium bicarbonate solution, unless you're referring to its use as a component in the eosin dye.


The acid alcohol is used between the hematoxylin staining and the eosin staining. It's purpose is essentially to remove any non-specific hematoxylin stain (reference). Hematoxylin is acidophilic (hence its capacity to bind nuclear components such as ribonucleic acids & deoxyribonucleic acids), so if you wash the slide with a weak acid, the hematoxylin which is just "sitting around" unbound to an acidic cellular component will be washed away, making the colouration much cleaner since only the acidophilic cellular componenets will still have the blue stain on them.

On a more technical tack, it also keeps you from getting large quantities of hematoxylin in your eosin stain, thus allowing you to use your eosin stain for more slides before it no longer stains as well.

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