How are new staining protocols designed for microscopy? Is it manual work or does it rely on computer simulations?

Say for example I thought the H&E staining does not bring enough contrast. How would I go about producing The Superb H&E Staining Protocol (not necessarily using just hemalum and eosin Y)?

Looking this from a mathematical point of view, the staining is some operator O applied on image I to produce enhanced image E. While clearly the operator O depends on the particular type of specimen, what I want to see, etc., there are surely some general principles guiding the search for finding an appropriate operator O? What are they?

Since the question is on hold, maybe I can try to put it even more clearly. How was the H&E staining invented? What was the process for its invention? I don't see why it would be unclear what I am asking. Dear biologists please take a more interdisciplinary approach.. If the question really is not answerable as such, please explain why. There's probably an answer in that.

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    $\begingroup$ About what new staining protocols are you thinking? And what kind of simulations do you think of? $\endgroup$
    – Chris
    Jul 27, 2014 at 15:40
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    $\begingroup$ Added example to the question. $\endgroup$
    – micro
    Jul 28, 2014 at 15:13
  • $\begingroup$ Put on hold now after I added the requested example? Duh. $\endgroup$
    – micro
    Jul 28, 2014 at 19:12
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    $\begingroup$ The question is what you want to stain and how. A newer method would be fluorescence micropscopy with specific antibodies coupled to fluorescence dyes. You can also couple the dyes to proteins which bind to cell surfaces. $\endgroup$
    – Chris
    Jul 28, 2014 at 19:34
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    $\begingroup$ As far as i understand staining, you have to primarily know what it is that you want to observe since most of the time, the sample of interest can not be seen unaided. Once you determined, what the nature of the sample is i.e. protein, or lipid or a specific structure, then you think of molecules/dyes that are known to interact/bind to it, preferably in a specific manner, providing a good contrast against the background. For example for staining DNA, you use DAPI. So its highly dependent on the nature of the molecule/structure you want to observe! $\endgroup$ Jul 28, 2014 at 20:52

1 Answer 1


This answer addresses the question of how stains were developed historically, which may bring into perspective your overly modern assumptions.

Most of these older (but stil very usefull!) staining techniques, such as haematoxylin (or rather haematein), cochenial/carmine etc. were inspired by the work of textile dyers of that era, starting somewhere in the middle of the 19th century.

It was more a question of trial and error, coïncidence and luck, rather than it being a case of systematic, structured research, starting from the well known molecular structure of the dye and the sample to be stained... Most of the time those were still -at least partial- mysteries.

You should keep in mind, that many of the protocols still in use, were developed in an era when the dye industry as well was still in its infancy, so to speak. Standardization of dyes, Biological Stain Commission and all that, came later. Biologists, histologists etc. used what worked for them. At that time, at that place, with that lot of dry dye powder. Much inspiration, the right idea at the right time and a fair amount of luck. Up to well into the 20th century.

As an illustration: in 1917-1918 there was a shortage on haematoxylin in the German histopathological labs, due to the allied blockade. A lab worker, Fraulein Wilhelmina Schmidt, inspired by the impossibillity to remove elderberry stains from her hands, wondered if it would be possible to use elderbery juice as a substitute for haematoxylin. As there wasn't really an alternative, it was tried. Elderbery juice was concentrated by boiling, diluted with an alum solution and tried. It turned out to work very well.

The story went unnoticed until 1949, when an article on it was published in the German “Zeitschrift für die Wissenshaftliche Mikroskopie”. In the article, the author, Grüber, pointed out that, even after more than 20 years of storage, the slides were still vividly stained, not showing any trace of fading.

  • $\begingroup$ I think this is a useful answer, but I have edited the first sentence as it seems to me that site policy does not allow answers that explicitly claim not to be answers. $\endgroup$
    – David
    Apr 22, 2017 at 7:55
  • $\begingroup$ I have noticed that indigo dye doesn't seem to dissolve in anything. Wonder if that would make a usable stain, or if it would totally stain everything blue. But the natural precursor to indigo is a glycoslyated molecule, similar to X-gal, maybe the precursor could be used in a manner similar to beta-galactosidase staining. $\endgroup$
    – user137
    Apr 22, 2017 at 18:16
  • $\begingroup$ Do you mean indigo carmine, CI 73015 (synthetic)/ CI 75781 (natural)? $\endgroup$
    – guest
    Apr 22, 2017 at 21:55

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