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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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Periodic Acid Schiff (PAS) Stain Protocol

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

  1. De-paraffin slides in xylene (1) —————— 2 minutes (re-use)
  2. De-paraffin slides in xylene (2)—————— 2 minutes (re-use)
  3. Clear slides in 100% alcohol——————— 2 minutes
  4. Clear slides in 100% alcohol——————— 2 minutes
  5. Hydrate slides in 95% alcohol——————- 2 minutes
  6. Hydrate slides in running water—————– 5 minutes
  7. 0.5% periodic acid ——————————– 5 minutes (re-use)
  8. Schiff’s reagent at 37C—————————- 15 minutes (re-use)
  9. Wash in running water ————————— 10 minutes
  10. If staining for glycogen, Hematoxylin ——— 20 seconds (re-use)
  11. If staining for fungus, light green ————— 30 seconds (re-use)
  12. Dehydrate in 95% alcohol———————— 30 seconds
  13. Dehydrate in 100% alcohol———————- 1 minute
  14. Dehydrate in 100% alcohol———————- 2 minutes
  15. Dehydrate in 100% alcohol———————- 2 minutes
  16. Clear in xylene (3)——————————— 2 minutes (re-use)
  17. Clear in xylene (4)——————————— 5 minutes (re-use)

Results:           Hematoxylin – Glycogen, mucin, fibrin, or thrombi, colloid droplets, hyaline of arteriosclerosis, hyaline deposits in glomeruli, granular cells in the renal arterioles where preserved, most basement membranes, colloid of pituitary stalks and thyroid, amyloid infiltration may show a positive reaction – Rose to Purplish Red

                        Nuclei – Blue

Results:          Light Green – Fungi – Red                 Background – Pale Green

 

 

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Masson’s Trichrome Blue Protocol

Masson’ Trichrome Blue
Safety equipment: Work under a hood with lab coat, gloves, and glasses.
1. De-paraffin slides in xylene (1)———————- 2 minutes (re-use)
2. De-paraffin slides in xylene (2)———————- 2 minutes (re-use)
3. Clear slides in 100% alcohol————————– 2 minutes
4. Clear slides in 100% alcohol————————– 2 minutes
5. Hydrate slides in 95% alcohol————————- 2 minutes
6. Hydrate slides in running water———————– 5 minutes
7. Bouins at 60 degrees Celsius————————– 1 hour (re-use)
8. Wash in running water ——————————– 10 minutes
9. Weigert’s Hematoxylin (make fresh) —————— 10 minutes (dump)
10. Wash in running water ——————————– 10 minutes
11. Biebrich Scarlet Acid Fuchsin————————- 10 minutes (re-use)
12. Rinse in running water——————————– 2 minutes
13. Phosphomolybdic / phosphotungstic acid—————- 10 minutes (re-use)
14. No rinse, goes directly into blue
15. Analine Blue—————————————— 10 minutes (re-use)
16. Rinse in running water——————————– 1-2 minutes
17. 1% Glacial Acetic Acid——————————– 5 minutes (re-use)
18. Dehydrate in 95% alcohol—————————— 1 minute
19. Dehydrate in 95% alcohol—————————— 1 minute
20. Dehydrate in 100% alcohol—————————– 1 minute
21. Dehydrate in 100% alcohol—————————– 2 minutes
22. Dehydrate in 100% alcohol—————————– 2 minutes
23. Clear in xylene (3)———————————– 2 minutes (re-use)
24. Clear in xylene (4)———————————– 5 minutes (re-use)

Results:
Nuclei- Black
Cytoplasm, keratin, muscle fiber and intercellular fibers- Red
Collagen- Blue