Trichrome Stain

Chromotrope 2R 0.6gm, Aniline Blue 0.3gm,

Glacial Acetic Acid 1.0 ml, Phosphotungstic acid 0.8gm,

distilled water 100 ml.

Fixation : 10%NBF

1. Deparafinize sections through xylene.
2. Hydrate tissues through graded ethanol.
3. Rinse sections in distilled water.
4. Place in Bouin's solution (at 58 degrees C) for 10 minutes.
5. Rinse in running water until the yellow fades from the sections.
6. Place sections in Weigert's iron hematoxylin solution for 5 minutes. (see Acid Fuchsin Van Gieson Stain)
7. Rinse sections in running tap water for 10 minutes.
8. Place sections in Trichrome stain for 15 minutes.
9. Place in 0.5% acetic acid water 2 minutes, 2 changes.
10. Deydrate in graded alcohols..
11. Clear through xylene and mount with a synthetic resin medium.

Muscle Fibers - red

Collagen - blue

Nuclei - blue/black

Erythrocytes - red

0.5 % Acid Fuchsin

0.5 gm of Acid Fuchsin in 100 ml distilled water

Aniline Blue Solution

0.5 gm Aniline Blue, 2 gm Orange G, 1gm phosphotungstic acid, dissolve in 100 ml distilled water.

Fixation: Zenker

1. Deparafinize sections through xylene.

2. Hydrate tissues through graded ethanol.

3. Rinse sections in distilled water.

4. Dezenkerize if sections are Zenker fixed (10 minutes iodine, 10 minutes hypo solution).

5. Rinse in distilled water.

6. Stain sections in Acid Fuchsin solution for 1-5minutes depending on intensity of stain desired.

7. Wipe off excess dye from slides and immerse slides in the Aniline Blue solution for 20minutes to 1 hour.

8. Transfer to 95% ethanol, several changes to remove excess dye.

9. Dehydrate clear and mount with a synthetic resin.

Collagenous fibers - blue

Nuclei - red

neuroglia fibers - red

myelin - yellow

ground substances of cartilage, bone, mucus, amyloid - blue