Massson’s trichrome staining

[Seung Jae Jeong]

Massson’s trichrome staining

12. 7. 2001. by eun-jung

Materials

 

Bouin’s solution (Sigma, HT 10-1-32, 1L)

Weigert iron hematoxylin solution (Sigma, HT 10-79, A-B set)

Biebrich scarlet (Sigma, B-6008, 25g, 55%)

–          1% stock solution = 9.091g / 500ml

Acid fuchsin (Sigma, A-3908, 25g, 85%)

–          1% stock solution = 1.18g / 100ml

Phosphomolybdic acid, PMA (Sigma, P-0550, 25g, ACS grade)

–          100 x stock solution = 0.5g / 100ml

Phosphotungstic acid, PTA (Aldrich, 22420-0, 25g)

–          100 x stock solution = 0.5g / 100ml

Aniline blue (Fluka, 95290, 25g)

–          100 x stock solution = 0.5g / 100ml

Acetic acid, glacial

DDW

 

Working solution

	Stock solution		Acetic acid	DDW	etc.
Iron hematoxylin	A	25ml			Valid 7days
	B	25ml
Biebrich scarlet -acid fuchsin	BS	45ml	500ul	0
	AF	5ml
Phosphomolybdic –phosphotungstic acid	PMA	250ul	0	49.5ml	Dispose after a use
	PTA	250ul
Aniline blue	aniline	500ul	1ml	49.5ml
0.5% acetic acid			250ul	49.75ml	Dispose after a use

Procedure

 

1.      Fix in Bouin’s solution.

2.      Embed with paraffin.

3.      Section into a thickness of 6mm.

4.      Stain below procedure

Xylene I, II, III, IX  ————————————————- each 10min.

Aceton —————————————————————— 2-3times dipping

100% ~ 70% EtOH—————————————————- 5-6times dipping

Tap water and DDW washing

Bouin’s solution ————————-pre-warmed and incubation at 56°C for 1hr

or at RT for overnight

While incubation time, make the stain solutions

Tap water washing until yellow is discolored

DDW washing

Weigert iron hematoxylin ——————————————– 10min.

Tap water washing —————————————————- 10min.

DDW washing

Biebrich scarlet-acid fuchsin —————————————- 1min.

Tap water and DDW washing

Phosphomolybdic phosphotungstic acid ————————— 10-15min.

(DDW washing)

Aniline blue ———————————————————— 20min.

Tap water and DDW washing

0.5% glacial acetic acid  ——————————————– 3-5min

DDW washing

90%, 95%, 100%, 100% EtOH ————————————- 5-6 times dipping

Xylene I, II, III, IX ————————————————— each 10min.

Mount with Canada balsam

 

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