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Hematoxylin & Eosin Staining

microscopic bone marrow stained with H&E

As you can see by our pictures the routine hematoxylin and eosin (H&E) stain is a most popular choice for research, clinical, and vet pathologists.  Before immunohistochemistry (IHC) and immunofluorescence (IF), pathologists would rely solely on routine and special staining for their tissue diagnosis.

Can you diagnosis the correct answer below?

 

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6 Recipes for Hematoxylin

There are many varieties and uses of hematoxylin.  It has been in use for over a 160 years.  The perhaps best well-known is the use of the hematoxylin and Eosin (H&E) staining procedure.  For those who do not already know, the H&E is a routine satin done as the first stain on all projects to understand the basic morphology of the tissue you’re looking at.  The hematoxylin in this stain is used to identify the nuclei of all types of tissues.  Hematoxylin can be used in two different methods.  There is the Progressive method, where the staining is done by creating a hematoxylin solution which has an excess of salts or acid.  This increases the selectivity for nuclei.  The regressive staining involves over staining of the tissues in a relatively neutral hematoxylin.  The slides are then brought through an acid wash, which takes off excess stain followed by an base wash to neutralize the the acid process.  This is more difficult than it looks and takes practice.  The hematoxylin recipes here are most of the most common ones used although most labs tend to buy pre-made kits.  Our company makes the Weigert’s hematoxylin from scratch but choose to buy the Harris hematoxylin.  This is because, the mercuric chloride used to make Harris hematoxylin is not now available in the US otherwise we would make it.

We now have a hematoxylin page.  Here you can see and download all the most used hematoxylin recipes.

http://histologistics.com/histology/6-recipes-for-hematoxylin/

 

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Hematoxylin and Eosin Quality Control

We need to ensure quality of H&E sections, so when reviewing the daily H&E, we are looking for blue nuclei with chromatin and a well stained nuclear membrane. Also we are looking for 3 shades of eosin, light, medium and dark red/pink. Some of this is determined by the kind of eosin used.  If a control is questionable we should run a second control that we are sure of to ensure that the problem, is a staining problem and not a tissue problem. Over decalcified, over heated or poorly processed/fixed tissue can stain poorly even if all solutions in the H&E stain are OK. When doing H&E on frozen sections extra care must be taken if the slides are fixed in Formalin after cutting. Formalin or concentrated formaldehyde can act as a reducing agent. The best way to remedy this problem is to change the hematoxylin frequently. Frequently can be defined by the lab.  Some labs like clinical hospitals, where the volume is high, may determine a frequency based on number of slides, day or weeks.  Clinical labs are usually controlled by the pathologist, therefore whatever the pathologist wants, is what will become the norm. For smaller labs this is not the case.  Generally a smaller lab will inspect the color intensity of the hematoxylin on a weekly basis to determine the right time to change it.  As a rule of thumb, hematoxylin must be filtered before each days use.  This helps to keep the floating precipitate out, removes any extraneous cells and lets it keep longer.  The time in the 95% alcohols after eosin is where the three shades of eosin are differentiated so these times may need to be adjusted for best results.

Rat Liver H&E 20x
Rat Liver H&E 20x

reducing agent (also called a reductant or reducer) is the element or compound in an oxidation-reduction reaction that donates an electron to another species. Because the reducing agent is losing electrons, we say it has been oxidized. (http://en.wikipedia.org/wiki/Reducing_agent)

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Hematoxylin in Histology

Epididymis H&E 20xHematoxylin, the most commonly used nuclear dye, most commonly used natural dye, extracted from heartwood of the logwood tree which is native to Central America, made in USA! 

Considering all of its versatility in routine and rare variations of Hematoxylin staining, it can stain the following;

Nuclei, mitotic structure, mitochondria, mucin, hemoglobin, elastic fibers, muscle, collagen, axons, phospholipids, protozoa, fatty acids, myelin sheath, alpha and beta cells of the pituitary, pancreatic islets and also certain types of metal.

Oxidants and Mordents-

Hematein is the oxidation product of hematoxylin.

The conversion of hematoxylin to hematein is a process known as ripening which may be achieved naturally through exposure to air (Delafields hematoxylin).

There are also chemical oxidants to hasten the ripening process, mercuric oxide, sodium iodate and potassium permanganate.

PH will have effect on rate of oxidation.

A neutral aqueous solution of hematoxylin will form hematein in a few hours.

Alkaline solutions will affect for a more rapid oxidizing process.

Acid solutions will affect for a more slow oxidizing process.

Mordents- act as a link from the dye to the tissue. Aluminum and Iron are most commonly used, in some cases the mordant is also the oxidizer. For a stable solution the mordant chosen must be a non oxidizer, such as ammonium alum, phosphotungstic acid and phosphomolybdic acid. For a rapidly oxidizing short-lived and unstable solutions that only last up to 24 hours, mordents such as ferric chloride in Wiegerts hematoxylin and also ferric acetate or ferric alum are used.

The combination of mordant and Dye is called a “lake”, in the hematein mordant combination lake there is a positive charge that functions as a cationic or basic dye, this is sometimes called a basophilic stain.

MORE TO COME, READ ABOUT EOSIN AND THE COMBINATION