006005004137.GOT
Date: 03-JAN-2023
Author: IB. Version: 2

Trichrome Staining Protocol
Validated for all CELLINK bioinks, including alginate, nanofibrillated cellulose,collagen and GelMA based bioinks. This is a suggested procedure, please adjust
according to your experimental needs.

Protocol aim

Materials needed

The aim of this protocol is to provide instructions fortrichrome staining of sectioned paraffin embeddedconstructs. Follow Fixation Protocol, Paraffin
Embedding Protocol and Sectioning Protocol before
starting this protocol.

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Microscope slides with sectioned construct
Recommended thickness: 8-12 µm
Beakers for microscope slides
Distilled water
96% Ethanol
100% Ethanol
Xylene or xylene substitute, e.g. Shandon
Xylene Substitute (Thermofisher, Ref: 9990505)
Microscope slide box
56°C oven
Trichrome staining kit, Abcam Cat. No.:
ab150686
Mounting medium, e.g. Vector Laboratories
Cat.No.:H-5000
Cover glass

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Protocol
All handling and use of dyes, ethanol and xylene/xylene substitute must be done inside a fume hood with proper
PPE and disposed according to local regulation. If using another Trichrome Staining kit adjust the protocol
accordingly.

1.

Deparaffination and rehydration

MATERIAL
Microscope slides with sectioned construct
Xylene or xylene substitute
100% Ethanol
96% Ethanol
Distilled water

DESCRIPTION•

Deparaffinize and rehydrate sections by moving microscope slides with sectioned construct throughfollowing series:
1.
2.
3.
4.

2.

Xylene or xylene substitute: 3 x 5 min
100% ethanol: 1 min
96% ethanol: 1 min
Distilled water: at least 2 min

Trichrome stain, Bouin’s Fluid

MATERIAL
Microscope slide box
Bouin’s FluidOven at 56°C

DESCRIPTION••••••

Make a humified chamber of the microscope box by adding wet paper at the bottom.
Blot of the samples and place the microscope slides horizontally in the humified chamber.
Add Bouin’s Fluid to the sections, close the box and carefully transfer the box to the oven.
Incubate 60 min followed by a 10 min cooling period.
Rinse in tap water until sections are completely clear.
Rinse once in distilled water.

Note: Bouin’s Fluid is toxic, keep lid properly closed whenever not within a fume hood and handle withoutermost care.

3.

Trichrome stain, continued

MATERIAL
Trichrome staining kit
96% Ethanol

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DESCRIPTION•

Apply the staining solutions in following order, always adding enough to completely cover the sections:
1. Mix equal amounts of Weigert’s (A) and Weigert’s (B) to maintain working solution of Weigert’s
Iron Haematoxylin: 5 min.
2. Rinse in tap water until sections are completely clear.
3. Rinse once in distilled water.
4. Biebrich Scarlet/Acid Fuchsin Solution: 15 min.
5. Rinse in distilled water.
6. Phosphomolybdic/Phosphotungstic Acid: 15 min.
7. Remove Phosphomolybdic/Phosphotungstic Acid but do not rinse.
8. Aniline Blue solution: 7 min.
9. Rinse in distilled water.
10. Acetic acid solution (1%): 3-5 min.
11. Dehydrate slides quickly in two changes of 96% ethanol.

Note: Blot off excess solution between the steps through tapping the slide (on the side, i.e. so it is held
vertically) towards a paper covered area of the bench. Especially important after the washes since excesswater/ethanol will dilute the staining solutions if not removed.

4.

Dehydration and clearing

MATERIAL
100% Ethanol
Xylene or xylene substitute

DESCRIPTION•

Dehydrate and clear slides by moving the stained microscope slides through following series:
1.
2.

5.

100% ethanol: 2 x 1 min
Xylene or xylene substitute: 3 x 1 min

Mount and coverslip

MATERIAL
Mounting medium
Cover glass

DESCRIPTION•••

Apply a drop of mounting medium to the stained slides.
Cover with a cover glass, apply carefully to avoid air bubbles.
Let air dry horizontally overnight.

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