MASSONS Trichrome Principle

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SURGICAL PATHOLOGY - HISTOLOGY Dat e:

STAINING MANUAL - CONNECTIVE TISSUE Page: 1 of 4

COLLA GEN - MA SSON'S T RI CHROME STA I N(T RI )

PURPOSE: Used t o diff erent iat e between collagen and smooth muscle in

t umors, and the increase of collagen in diseases such as cirrhosis.Rout ine st ain f or liver and kidney biopsies.

PRI NCI PLE: As t he name implies, t hree dyes are employed select ively

staining muscle, collagen f ibers, fibrin, and eryt hrocyt es. The general rule

in t richrome staining is that t he less porous t issues are colored by t he

smallest dye molecule; whenever a dye of large molecular size is able to

penet rate, it will always do so at t he expense of t he smaller molecule.

Ot hers suggest t hat t he t issue is st ained first wit h t he acid dye, Biebrich

Scarlet , which binds with t he acidophilic t issue components. Then when

treated with the phospho acids, the less permeable components retain the

red, while t he red is pulled out of t he collagen. At t he same t ime causing a

link wit h t he collagen t o bind wit h t he anil ine blue.

FIXATIVE: Bouin's is preferred, 10% formalin.

TECHNI QUE: Cut paraf f in sect ions 4 m

EQUI PMENT: Rinse glassware in DI water. Coplin jars, 60°C oven or

wat erbat h, microwave

REAGENTS:

Boui n ’ s Fi x at i v e

Sat u rat e d picr ic acid 1 50 0.0 ml

Formaldehyde 500.0 ml

Glacial acet ic ac id 100.0 ml

Mix well, label, dat e and init ial.

Stable for 2 years.

CA UT I ON: Ca r c i n og en i c , i r r i t a nt .

Bi ebr i c h Sc ar l et :

Biebr ich scar let 2.7 gm

Acid f uchsin 0.3 gm

Dist i l led wat er 300.0 ml

Glacial acet ic acid 3 .0 ml

Mix well, label wit h init ial and

date. Solut ion is stable for 6months.

CAUTI ON: Avoi d cont ac t and i nha l a t i on.

CONNECTIVE TISSUE

MA SSON’ S TRI CHROME Page: 2 of 4

Wei ger t ' s I r on Hem at ox y l i n

St o c k So l u t i o n A :

Hemat oxy lin 5.0 gm

95% alcohol 500.0 ml

date. St able for 1 year.

CAUTI ON: Fl ammab l e , avo i d cont ac t and

i n h a l a t i o n .

St o c k So l u t i o n B:

2 9 % f err ic chlor ide 20 .0 ml

Dist i l led wat er 475.0 ml

Hydrochloric acid 5 .0 ml

CAUTI ON: Co r r os i ve , avo i d con t ac t and

i n h a l a t i o n .

Wei ger t ' s Hem at ox y l i n

Wo r k i n g So l u t i o n

Solut ion A 25 .0 ml

Solut ion B 25 .0 ml

Mix well, solut ion will remain

stable for 3 - 4 days.

Ph os p ho t u ng s t i c /

Ph os p ho m ol y b di c A c i d So l u t i o n:

Phosphotungst ic acid 25 .0 gm

Phosphomolybdic acid 25.0 gm

Dist i l led wat er 1000 .0 ml

date. Solut ion is st able for 6

months.

CAUTI ON: Avoi d con t ac t and i nha l a t i on.

A ni l i ne Bl ue:

Anil ine b lue 2.5 gm

Glacial acet ic ac id 1 .0 ml

months.

Ca ut i o n: A v o i d c o n t a c t a nd i n ha l a t i o n.

1 % A c e t i c A c i d :

Glacial acet ic ac id 10 .0 mlDist i l led wat er 1000 .0 ml

dat e. Solut ion is st able f or 1 y ear.

Ca ut i o n: A v o i d c o n t a c t a nd i n ha l a t i o n.

SAFETY: Wear gloves, goggles and lab coat . Avoid cont act and inhalat ion

of dyes and chemicals.

Bouin's cont ains: formaldehyde, a known carcinogen, picric acid canbecome explosive when dry. Toxic by skin absorbt ion. Keep hot uncapped

Bouin’s under t he hood.

Phosphomolybdic, phosphotungsic acid powders, and acet ic acid solut ions

are skin and eye irrit ant s, and st rong corr osives.

CONNECTIVE TISSUE

PROCEDURE:

1. *Mordant in Bouin's solut ion, microwave 1 minute, allow to stand 15

minut es.

2. Wash in running t ap wat er to remove t he p icr ic ac id, 5 minutes.

3 . Weiger t ' s work ing hemat oxy lin , 10 minut es.

4 . Blue in running t ap wat er fo r 5 minutes , r inse in d ist i lled wat er.

5 . Biebr ich scar le t fo r 5 minu tes.

6 . Rinse in d ist il led wat er .

7. Phosphotungst ic/ phosphomolybdic acid for 10 minut es, discard

solut ion.

8 . Transfer di rect ly int o Ani line blue for 5 minutes .

9 . Rinse in d ist il led wat er .

10. 1% Acet ic acid for 1 minut e, discard solut ion, r inse in dist i lled wat er.11. Dehydrat e, clear, and coverslip.

*Convent ional met hod: Mordant in Bouin's solut ion, 60 °C f or 1 hour.

RESULTS:

Nuclei b lack

Cy t oplasm , muscle, ery t hrocy t es red

Collagen blue

NOTES:

1. Light green may be subst i tut ed for Ani line blue..

2. 5% phosphotungst ic acid for 5 minut es, must be subst itut ed when

using Light green.

3. When st aining liver biopsies, t he collagen is bett er light blue t han

dark blue.

REFERENCE:

Sheehan D, Hrapchak B, Theory and pract ice of Histot echnology, 2nd Ed,

19 80 , pp 18 9-1 90 , Batt elle Press, Ohio

Bancrof t J, St evens A, Theory and Pract ice of Histological Techniques, 2ndEd, 19 82 , pp 1 31 -13 5, Churchill-Livingst on, NY

Luna L, Manual of Histologic St aining Met hods of t he AFIP, 3rd Ed, 1968, pp

9 4 -9 5 , McGraw-Hil l, NY

Carson F, Histot echnology A Self-Inst ruct ional Text , 1st Ed, 19 90, pp

1 42 -1 4 3 , ASCP, I ll

CONNECTIVE TISSUE

Crookham,J, Dapson,R, Hazardous Chemicals in t he Hist opat hology

Laboratory, 2nd ED, 19 91, Anatech

Prepared: By:

Approved: By:Downloaded from WebPath: Internet Pathology Laboratory

htt p:/ / www-medlib.med.ut ah.edu/ WebPath/ webpat h.ht ml

PROCEDURE CARD

TRI CHROME STA I N, MA SSON'S ( TRI ) - COLLA GEN Page 1 of 2

PROCEDURE:1. *Mordant in Bouin's solut ion, microwave 1 minut e, allow to stand 15 minutes.

2. Wash in running tap wat er t o remove t he picric acid, 5 minutes.

3. Weigert ' s work ing hematoxy lin , 10 minutes.4. Blue in running tap wat er for 5 minut es, rinse in dist i lled wat er.

5 . Biebr ich scar le t f or 5 minu tes.

6 . Rinse in dist i lled wat e r.

7. Phosphotungst ic/ phosphomolybdic acid for 10 minut es, discard solut ion.

8. Transfer di rect ly into Anil ine b lue for 5 minutes.

9 . Rinse in dist i lled wat e r.

10. 1% Acetic acid for 1 minute, discard solution, r inse in dist i l led wat er.

11. Dehydrat e, clear, and coverslip.

*Convent ional met hod: Mordant in Bouin's solut ion, 60 °C fo r 1 hour.

RESULTS:Nuclei black

Cytoplasm, muscle, erythrocytes red

Collagen blue

SAFETY/ PPE: Wear gloves, goggles and lab coat. Keep hot Bouin’s under t he fume hood.

We i g e r t ' s I r o n He m at o x y l i n

St o ck S ol u t i o n A :

Hemat oxylin 5.0 gm

95% alcohol 50 0 .0 ml

Mix well, label wit h init ial and date. Stablefor 1 year.

CAUTION: Fl amm abl e

St o c k So l u t i o n B :

2 9 % f erric chlor ide 2 0 .0 ml

Dist illed wat er 47 5 .0 ml

Mix well, label wit h init ial and date. Stable

for 1 year.

Wo r k i n g So l u t i o n

Solut ion A 2 5 .0 ml

Solut ion B 2 5 .0 ml

Mix well, solution will remain stable for 3 -

4 days.

Bo ui n ’ s Fi x a t i v e

CA UT I ON: Ca r c i n o ge ni c , i r r i t a n t ,

t o x i c .

B i e br i c h Sc a r l e t :

Biebrich scarlet 2.7 gm

Acid fuchsin 0.3 gm

Mix well, label with init ial and date. Solut ion

is st able for 6 mont hs.

CAUTI ON: Poss i b l e ca r c i nogen

Ph os p ho t u ng s t i c /

Ph os p ho m ol y b di c A c i d So l u t i o n:

Phosphotungst ic acid 25 .0 gm

Dist illed wat er 1 0 00 .0 ml

Mix well, label with init ial and date. Solut ion

PROCEDURE CARD

TRI CHROME STA I N, MA SSON'S ( TRI ) - COLLA GEN Page 2 of 2A n i l i n e B l u e :

Aniline blue 2.5 gm

Glacial acet ic acid 1 .0 mlMix well, label with init ial and date. Solution

1 % A c e t i c A c i d :

Glacial acet ic acid 1 0 .0 ml

Dist illed wat er 1 00 0 .0 ml

Mix well, label with init ial and date. Solutionis st able for 1 year.

1 % ACETI C A CI D:

date. Solution is st able f or 1 year.

Ca ut i o n: Sk i n an d e y e i r r i t a nt

EXPIRATION:

A NI LI NE BLUE:Aniline b lue 2.5 gm

date. Solut ion is stable for 6

months.

Ca ut i o n: A v o i d c on t a c t a nd i n h al a t i o n.

EXPIRATION:

ANI LI NE BLUE

BI EBRI CH SCARLET:

Biebr ich scar let 2.7 gm

Acid f uchsin 0.3 gm

months.

EXPIRATION:

BI EBRI CH SCARLET:

PHOSPHOTUNGST I C/

PHOSPHOMOLYBDI C A CI D

SOLUT I ON:

Phosphot ungst ic acid 25 .0 gm

date. Solut ion is stable for 6

months.

EXPIRATION:

PHOPHOTUNGST I C /

PHOSPHOMOLYBDI C A CI D:

WEI GERT' S I RON HEMAT OXYLI N:

St o c k So l u t i o n A :

Hemat oxy lin 5.0 gm

95% alcohol 100 .0 ml

Mix well, label wit h init ial anddate. St able for 1 year.

CAUTION: FLAMMABLE, avoid

cont act and inhalat ion

EXPIRATION:

WEI GERT’ S I RON HEMAT OXYLI N:

St o c k So l u t i o n B:

2 9 % f err ic chlor ide 20 .0 ml

CAUTI ON: Co r r os i ve , avo i d con t ac t and

i n ha l a t i o n.

EXPIRATION:

WEI GERT' S HEMAT OXYLI N

MASSONS Trichrome Principle

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