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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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Eosin in Histology

BoneMarrow_H&E_x10

Most frequently this is an anionic stain, and is most commonly used as the counterstain.

When used properly three shades of pink can be obtained with eosin alone. Erythrocytes, collagen and cytoplasm, muscle or epithelial cells should stain different shades or intensities of pink. Erythrocytes and eosinophilic granules are bright pink to red, cytoplasm and other tissue elements are various shades of pink.

Pale staining with eosin usually results from the PH being over 5.0, it should be from 4.5-5.0. It also may result if the sections are very thin, if they are dehydrated to long, or if they are allowed to stay in the lower concentration alcohols because the water is what decolorizes eosin (that is the reason the step after eosin in any H&E protocol is always 95% alcohol).

If the cytoplasm is over stained it may be because the sections were stained for too long, the eosin may also be too concentrated. The sections may be too thick, or the tissue may have been dehydrated to quickly.

If the eosin staining is not well differentiated (you are not able to see three shades of pink) the slides may need to be rehydrated and then dehydrated again, to expose the eosin to water a second time.

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What is Tissue Histology?

Tissue histology is tissue morphology. This means that a histologist will examine all tissue samples grossly. All tissue has a definite “normal” appearance as seen inside and outside its normal environment. This tissue must be described as color, shape, and size before it can be processed for microscopic evaluation. This description (morphology) is part of the tissue histology. The second part is the microscopic description (morphology) of the tissue after it has been stained. The typical stain done on all tissues is the hematoxylin and eosin (H&E). This shows the basic structure of the tissue. All tissue components that are basophilic are stained with eosin and will have a pink hue. All the tissue components that are acidophilic are stained purplish blue with the hematoxylin. These are typically the nuclei that turn blue. This stain allows the identification of a particular tissue type, (muscle, lung, and so on). The examination and description of the microscopic structures constitutes the tissue histology.