Protocol
Koneff's Trichrome
for Pituitary Cells
Materials
Solution A
| Material | Amount | |
|---|---|---|
| Aniline | 0.1 | mL |
| Ethanol, 90% | 100 | mL |
Solution B
| Material | Amount | |
|---|---|---|
| Acetic acid, glacial | 1 | mL |
| Ethanol, 90% | 100 | mL |
Solution C
| Material | Amount | |
|---|---|---|
| Azocarmine | 1 | g |
| Acetic acid, glacial | 1 | g |
| Distilled water | 100 | mL |
Solution D
| Material | Amount | |
|---|---|---|
| Aniline | 0.06 | mL |
| Ethanol, 90% | 100 | mL |
Solution E
| Material | Amount | |
|---|---|---|
| Phosphotungstic acid | 5 | g |
| Distilled water | 100 | mL |
Solution F
| Material | Amount | |
|---|---|---|
| Aniline blue | 0.5 | g |
| Orange G | 2 | g |
| Oxalic acid | 2 | g |
| Phosphotungstic acid | 0.05 | g |
| Distilled water | 100 | mL |
Solution G
| Material | Amount | |
|---|---|---|
| Acetic acid | 1 | mL |
| Distilled water | 100 | mL |
Tissue Sample
The instructions specify fixation in see Baley’s fixative. Other mercuric-chrome fixatives would probably be satisfactory. The instructions also specify 3-4 µ celloidin sections which have been attached to a slide, and from which the celloidin has been removed. Possibly, similar paraffin sections would be satisfactory.
Baley’s fixative consists of:
| Material | Amount | |
|---|---|---|
| Mercuric chloride | 3.4 | g |
| Potassium dichromate | 3.4 | g |
| Concentrated formalin (40%) | 25 | mL |
| Water | 225 | mL |
| Return to fixation instructions. | ||
Protocol
- Attach sections to slides and remove celloidin, or dewax sections with xylene and bring to ethanol.
- Although not specified, a water rinse followed by the iodine thiosulphate sequence and a second water wash, to remove mercury pigment would be advantageous at this point.
- Wash with 70% ethanol.
- Place into solution A for 45 minutes.
- Place into solution B for 1-2 minutes.
- Place into solution C for 2 hours at 56°C.
- Wash with water.
- Place into solution D until nuclei are red and the cytoplasm is pink.
- Place into solution B for 1-2 minutes.
- Place into solution E for 4 hours
- Place into solution F for 4 hours until basophils are blue.
- Return to solution E for 3-5 minutes.
- Wash with water.
- Rinse with solution G.
- Wash with water.
- Dehydrate with absolute ethanol.
- Clear with xylene and mount with a resinous medium.
Expected Results
- Nuclei – red
- Acidophils – orange-red
- Basophils – blue
- Chromophobes – pale grey
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- Gray, Peter. (1954)
The Microtomist’s Formulary and Guide.
Originally published by: The Blakiston Co.
Republished by: Robert E. Krieger Publishing Co.
Citing:
Koneff, (1938)
Stain Technology, v.13, pp.49
