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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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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

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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.

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Movat’s Modified Pentachrome Protocol

This is a quick version of Movat’s Pentachrome.  The long version is an overnight stain, I hate waiting till the next day to find out if the stain worked.  It is sometimes hard to visualize all 5 colors but with a aorta in the heart, it is possible.  The stain picture will have to be edited in later.

Control tissue that I use: Skin, Aorta in the heart

Safety equipment: Work under a hood with lab coat, gloves, and glasses.

  1. Deparaffinize and hydrate to DH2O
  2. Preheat Alkaline alcohol in 60C oven or water bath
  3. Mordant in bouin’s at 60C water bath——- 30 minutes (under hood!) (re-use)
  4. Cool—————————————– 10 minutes in room temp water
  5. Wash in running water—————– 10 minutes
  6. 1% Alcian blue————————— 25 minutes (re-use)
  7. Wash in running water—————– 5 minutes
  8. Alkaline alcohol at 60C —————- 10 minutes
  9. Wash in running water—————— 5 minutes
  10. Weigerts———————————– 15 minutes (dump)
  11. Differentiate in 2% ferric chloride for- 3 minutes (re-use)
  12. Rinse in H2O——————————- 10 seconds
  13. Sodium thiosulfate for——————– 1 minute (re-use)
  14. Wash in running water——————- 5 minutes
  15. Crocein scarlet/ Acid fuchsin———- 1.5 minutes (re-use)
  16. Wash in running water—————— 5 minutes
  17. 5% Phosphotungstic acid————– 5 minutes (re-use)
  18. Transfer directly to 1% acetic acid–  5 minutes (re-use)
  19. Wash in running water—————— 5 minutes
  20. Dehydrate quickly 95% alc. 1 min/2x    100% alc. 1 min
  21. Alcoholic saffron————————– 60 minutes (re-use)
  22. Rinse in 2 changes of 100% alcohol, 1 min each
  23. Clear in xylene and mount

Results:

Nuclei and elastic fibers—Black

Collagen—Yellow

Ground substance and mucin–Blue

Fibrinoid, fibrin–Intense Red

Muscle–Red

 

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