Masson's Trichrome Staining Protocol for FFPE spheroids and microtissues
Zbigniew Mikulski, Angela Denn
spheriod
microtissue
collagen
trichrome
FFPE
staining
quantitative
image analysis
autostainer
histology
Leica ST5020
Abstract
The following protocol has been optimized for image analysis-based quantification of collagen within FFPE spheroid samples. It is run on a Leica ST5020 automated stainer. This protocol can be adapted to manual staining.
PURPOSE:
Trichrome stains are used to differentiate collagen from other tissues. They are usually nuclear and cytoplasmic stains, followed by differentiation, then followed by a collagen fiber stain. Trichrome stains are useful for indicating fibrotic changes.
PRINCIPLE :
The staining reaction is based on the differential effect of acid dye on collagen and other tissue components.
Before start
Cut paraffin sections at 4µm.1. Slides must be completely dry before deparaffinzation.
- Bouin’s solution must be preheated to insure proper mordanting.
Steps
Deparaffinization Protocol
| A | B | C | D | E |
|---|---|---|---|---|
| Step | Reagent | Duration | Exact | Dip |
| 1 | Oven station | 5:00 | N | N |
| 2 | Oven station | 5:00 | N | N |
| 3 | Pro-par | 5:00 | N | Y |
| 4 | Pro-par | 5:00 | N | Y |
| 5 | Pro-par | 5:00 | N | Y |
| 6 | IPA 99% | 1:30 | N | Y |
| 7 | IPA 99% | 1:30 | N | Y |
| 8 | IPA 90% | 1:30 | N | Y |
| 9 | DI Water station | 2:00 | N | N |
Mordanting
For formalin fixed tissue, post-fix deparaffinized slides in Bouin’s solution for 1 hour at 56°C or at room temperature overnight. Do not leave in oven overnight. Bouin’s solution must be preheated to insure proper mordanting.1. Wash in water and discard first two changes to red container aqueous waste. Rinse in sink with running tap water for 5 - 10 minutes to remove the yellow color. Use warm water to remove excess yellow color.
- Rinse in sink with distilled water for 30 seconds.
Masson Trichrome Protocol MT3B for Leica ST5020 Autostainer
| A | B | C | D | E |
|---|---|---|---|---|
| Step | Reagent | Duration | Exact | Dip |
| 1 | Biebrich Scarlett | 5:00 | Y | Y |
| 2 | DI Water station | 3:00 | Y | N |
| 3 | PTA/PMA Working Solution | 5:00 | Y | Y |
| 4 | Aniline Blue | 5:00 | Y | N |
| 5 | DI Water station | 2:00 | Y | N |
| 6 | Acetic Acid, 1% | 2:00 | Y | Y |
| 7 | IPA, 99% | 0:20 | Y | Y |
| 8 | IPA, 99% | 0:20 | Y | Y |
| 9 | IPA, 99% | 1:00 | Y | Y |
| 10 | IPA, 99% | 1:00 | Y | Y |
| 11 | IPA, 99% | 1:00 | Y | Y |
| 12 | 50:50 Pro-par/IPA99 | 1:00 | Y | Y |
| 13 | Pro-Par | 3:00 | N | Y |
| 14 | Pro-Par | 3:00 | N | Y |
| 15 | Pro-Par |
Mounting
Use a resinous mounting medium to coverslip slides. Allow to dry flat for a minimum of 2 hours.
