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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


Nuclei and elastic fibers—Black


Ground substance and mucin–Blue

Fibrinoid, fibrin–Intense Red



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Glycosaminoglycans (GAGs)

What are the components of connective tissue?

They are ground substance, extracellular fibers and cellular elements. Ground substance is a matrix that allows diffusion and binds fibers and other tissue elements together.

Components of ground substance are glycoaminoglycans (GAGS), water, calcium.

4 functions of the ground substance are glue, lubrication, barrier, tensil strength.

What are the 2 groups of ground substance,  glycoaminoglycans (GAGS) and glycoproteins.

What are GAGS, they are structural proteins bound to protein core to create a bottle brush structure.

2 types of GAGS are hyaluronic acid and he sulfates.

They have a highly negative because of the sulfate group.

Explain how hyaluronic acid differs from the rest of the GAGS.

Longer chain and forms the base of the bottle brush that the core protein binds to.

Describe a proteoglycan monomer.

A bunch of GAGS around a central protein linked to a hyaluronic acid.

Components of ground substance are: sulfated glycoaminoglycans; heparin sulfate, dermatan sulfate, keratin sulfate, chondroitin sulfate (GAGS sulfated).

What are the function of gags.

They electrostatically bind to collagen for strength, trap water to make a gel that enables tissues to withstand compression.

They are a matrix for cells and blood vessels and a diffusion medium.

There are 5 electrolyte  molar concentrations to stain for. This week I stained for 3 of them. The alcian blue stain works well to detect it.

Every concentration had different results.
0.06 – Carboxylated and sulfated mucosubstances and mucins.
0.3 – Weakly and strongly sulfated mucosubstances and mucins.
0.5 – Strongly sulfated mucosubstances and mucins.
0.7 – Highly sulfated connective tissue mucosubstances.
0.9 Keratan sulfate.

Here are the results:

Rat colon 0.5 10x
Rat colon 0.5 10x
Rat Colon AB 0.7 10x
Rat Colon AB 0.7 10x
Rat colon AB 0.9 10x
Rat colon AB 0.9 10x
Rat knee joint AB 0.5
Rat knee joint AB 0.5