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

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