Mar 16, 2022
Masson's Trichrome Staining Protocol for FFPE spheroids and microtissues
- 1La Jolla Institute for Immunology
Protocol Citation: Angela Denn, Zbigniew Mikulski 2022. Masson's Trichrome Staining Protocol for FFPE spheroids and microtissues. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz82w5vx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: April 27, 2020
Last Modified: June 07, 2024
Protocol Integer ID: 36274
Keywords: 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.
Guidelines
- Decreased red staining usually indicates that the staining solution has aged or been overused and should be discarded. If blue staining of connective tissue appears faded, the section has probably been overdifferentiated in the acetic acid solution.
- Sections fixed in 10% neutral buffered formalin will stain poorly and unevenly if not mordanted in either Bouin or a mercuric chloride solution; however, mercuric fixatives should not be used because of the toxicity.
Materials
MATERIALS
Acetic acid GlacialFisher ScientificCatalog #A38-212
Pro-Par ClearantAnatech LTPCatalog #510
Trichrome Stain (Masson) KitSigma AldrichCatalog #HT15-1KT
Isopropyl Alcohol 99%Pacific Southwest Lab EquipmentCatalog #104B
Bouins FixativeMedical Chemical CorporationCatalog #456-A-2.5GL
Solutions and Reagents:
- Bouin’s Fixative (HT15-1KT)
- Aniline Blue Solution (HT15-1KT)
- Biebrich Scarlet-Acid Fuchsin Solution (HT15-1KT)
- Phosphomolybdic Acid Solution (HT15-1KT)
- Phosphotungstic Acid Solution (HT15-1KT)
- 1% Acetic Acid Solution
- Isopropyl Alcohol, 99%
- Pro-Par Clearant
Prepare Working Phosphotungstic/Phosphomolybdic Acid (PTA/PMA) Solution by mixing 1 volume of Phosphotungstic Acid Solution and 1 volume Phosphomolybdic Acid Solution with 2 volumes of deionized water.
Biebrich Scarlet-Acid Fuchsin and Aniline Blue solutions are ready to use.
Prepare a 1% acetic acid solution by adding 4ml of glacial acetic acid to 396ml of deionized water.
Equipment
ST5020
NAME
Automated Multistainer
TYPE
Leica
BRAND
Leica ST5020
SKU
LINK
Safety warnings
Bouin's Fixative contains picric acid. Picric acid containing less than 10% water is very explosive; therefore, it is important that solutions not be spilled in the oven and then allowed to evaporate. For this reason, the staining jar containing Bouin's Fixative/picric acid should be placed inside a secondary container while in the oven.
Before start
- Cut paraffin sections at 4µm.
- Slides must be completely dry before deparaffinzation.
- Bouin’s solution must be preheated to insure proper mordanting.
Deparaffinization Protocol
Deparaffinization Protocol
31m 30s
31m 30s
| 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
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.
- 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
Masson Trichrome Protocol MT3B for Leica ST5020 Autostainer
| 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
Mounting
- Use a resinous mounting medium to coverslip slides. Allow to dry flat for a minimum of 2 hours.