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Histology Stain Picro-sirius Red With Nuclei Stain

 

A comparison of the picrosirius red stain with and without a nuclear stain.

We took the original picrosirius red stain and modified it to create our version with crisp looking nuclei.

The picture on the left has been stained with Weigert’s hematoxylin and on the right, no nuclear stain.

Collagen – Red         Muscle – Green       Nuclei – black

 

histology stain picro-sirius red

What is your preference?

Collagen – the tissue glue, plays a vital role in maintaining structural integrity in tissue function.  This stain is used to detect many disease models such as liver fibrosis, skin wounds, myocardial scars, and arterial tissue.

 

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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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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

 

An Expert Histology Service

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Masson’s Trichrome Green Protocol

Masson’s Trichrome Green Protocol

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. Bouin’s Fixative (60 Celsius)———————- 30 minutes (re-use)
8. Wash in running water—————————— 5 minutes
9. Weigerts——————————————- 10 minutes (dump)
10. Wash in running water—————————— 5 minutes
11. Biebrich scarlet acid fuchsin———————- 5 minutes (re-use)
12. Rinse quickly————————————– 30-60 seconds
13. Phostung-Phosmoly acid (PTA-PMA)——————- 5 minutes (re-use)
14. No rinse
15. 2.5% Fast green———————————— 5 minutes (re-use)
16. Rinse quickly ————————————- 30 seconds
17. 1% Glacial acetic acid—————————– 2 minutes (re-use)
18. Dehydrate in 95% alcohol————————— 30 seconds
19. Dehydrate in 100% alcohol————————– 1 minute
20. Dehydrate in 100% alcohol————————– 2 minutes
21. Dehydrate in 100% alcohol————————– 2 minutes
22. Clear in xylene (3)——————————– 2 minutes (re-use)
23. Clear in xylene (4)——————————– 5 minutes (re-use)

Results:
Nuclei——————— Black to purple
Cytoplasm- —————- Shades of pink, red
Erythrocytes and myelin—- Scarlet
Collagen fibers———— Strong bluish-green
Mucus———————- Lighter green