Massons Trichrome Stain

2. MASSONS MASSONS
TRICHROME STAINTRICHROME STAIN
PRESENTED BY:PRESENTED BY:
MEHTAB SHAH MEHTAB SHAH
B.S MT 3B.S MT 3
RdRd
year year

(SDLS(LNH)(SDLS(LNH)

3. SPECIAL STAININGSPECIAL STAINING
Special stains are used to identify certain Special stains are used to identify certain
normal and abnormal substance present in normal and abnormal substance present in
the cells an tissue, which cannot be the cells an tissue, which cannot be
differentiated by routine haemotoxylin and differentiated by routine haemotoxylin and
eosin staining.eosin staining.

4. INTRODUCTIONINTRODUCTION
Masson's trichrome Masson's trichrome
stain is modified from stain is modified from
Masson in (1929).Masson in (1929).
Masson's trichromeMasson's trichrome
is a three-colour is a three-colour
stainingstaining protocol used protocol used
in in histologyhistology. .
This stainis used to This stainis used to
distinguishes between distinguishes between
collagen and smooth collagen and smooth
muscle in tumours.muscle in tumours.

5. MuscleMuscle
Muscle cells are specialized for contractility, and Muscle cells are specialized for contractility, and
they can be classified according to their they can be classified according to their
microscopic appearance into 3 categories: microscopic appearance into 3 categories:
Smooth muscleSmooth muscle which is found primarily in which is found primarily in
tubular organs. tubular organs.
Skeletal muscleSkeletal muscle which is generally attached to which is generally attached to
bones. bones.
Cardiac muscleCardiac muscle which is found in the wall of which is found in the wall of
the heart. the heart.

6. SMOOTH MUSCLESMOOTH MUSCLE
(involuntary)(involuntary)
Is developed from mesenchymal cells.Is developed from mesenchymal cells.
cell is spindle-shaped with an elongated centrally cell is spindle-shaped with an elongated centrally
located nucleus.located nucleus.
Microscopically; cells appear eosinophillic the Microscopically; cells appear eosinophillic the
presence of glycogen.presence of glycogen.
The cytoplasm contains bundles of myofilaments The cytoplasm contains bundles of myofilaments
called myofibrils.called myofibrils.
Surrounded by the sarcoplasm.Surrounded by the sarcoplasm.
The centrally situated nuclei with their fine The centrally situated nuclei with their fine
chromatin pattern stain with hemotoxylin and chromatin pattern stain with hemotoxylin and
have elongated appearance: they may contain have elongated appearance: they may contain
one or two nucleoli.one or two nucleoli.

7. COLLAGENCOLLAGEN
These connective tissue protein provide tensile These connective tissue protein provide tensile
strength.strength.
There are at least 27 types of collagen.There are at least 27 types of collagen.
Type 1,2,3.4 and 11 are the fibrillar collagens Type 1,2,3.4 and 11 are the fibrillar collagens
and most abundant.and most abundant.
The collagen of skin and bone is mostly type The collagen of skin and bone is mostly type
1,while cartilage collagen is mostly type 2.1,while cartilage collagen is mostly type 2.
Type 4 forms sheets instead of fibrils and is Type 4 forms sheets instead of fibrils and is
major collagen found basement membrane.major collagen found basement membrane.

8. ContdContd……………..……………..
Other collagen may form extra cellular Other collagen may form extra cellular
matrix meshwork, participate in cellular matrix meshwork, participate in cellular
anchorage.anchorage.

9. PRINCIPLEPRINCIPLE
Trichrome procedure are three dyesTrichrome procedure are three dyes
Sections are first stained with an acid dye Sections are first stained with an acid dye
such as Biebrich scarlet.such as Biebrich scarlet.
All acidophilic tissue elements such as All acidophilic tissue elements such as
cytoplasm. muscle and collagen will bind cytoplasm. muscle and collagen will bind
with the acid dyes.with the acid dyes.
The section are then treated with The section are then treated with
phosphotungstic and phosphomolydic phosphotungstic and phosphomolydic
acid.acid.

10. Contd…………………….Contd…………………….
Because cytoplasm is much less permeable than Because cytoplasm is much less permeable than
collagen the phosphotigstic and phophomolydic collagen the phosphotigstic and phophomolydic
acids cause the Biebrich scarlet to diffuse out of acids cause the Biebrich scarlet to diffuse out of
the collagen but not out of the cytoplasm.the collagen but not out of the cytoplasm.
Phosphotigstic and phosphomolydic have Phosphotigstic and phosphomolydic have
numerous acidic groups.numerous acidic groups.
Finally a dye of large particle size such as light Finally a dye of large particle size such as light
green is used that is able to enter collagen.green is used that is able to enter collagen.

11. FIXATIVEFIXATIVE
Bouin’s solution is preferredBouin’s solution is preferred
10% neutral buffered formalin 10% neutral buffered formalin
may be usedmay be used
TECHNIQUETECHNIQUE
Cut paraffin section at 4 micronsCut paraffin section at 4 microns

12. REAGENT REQUIRMENTREAGENT REQUIRMENT
Weigerts iron haematoxylinWeigerts iron haematoxylin
5% phosphomolydic acid5% phosphomolydic acid
5% phosphotungstic acid5% phosphotungstic acid
2% light green2% light green
Biebrich scarlet acid fushin solutionBiebrich scarlet acid fushin solution

13. PROCEDUREPROCEDURE
Depparaffinize section in xyleneDepparaffinize section in xylene
Clean xylene by alcoholClean xylene by alcohol

Rinse with distilled waterRinse with distilled water

weigerts iron hemotoxylin weigerts iron hemotoxylin
5-10 mint;5-10 mint;

14. Contd………………………Contd………………………
Rinse well in d/wRinse well in d/w

Biebrich scarlet acid fuchinBiebrich scarlet acid fuchin
5-10 mint;5-10 mint;
Rinse in d/wRinse in d/w
Phosphotungstic – phosphomolydic acid solutionPhosphotungstic – phosphomolydic acid solution
10-15 mint;10-15 mint;

15. Light green solutionLight green solution
2-5mint;2-5mint;
Dehydrate in absolute alcoholDehydrate in absolute alcohol
Clear in xyleneClear in xylene
Mount in synthetic resinMount in synthetic resin
MicroscopyMicroscopy

16. Quality control and safetyQuality control and safety
Practically every tissue has an internal Practically every tissue has an internal
control so no other control are needed, if control so no other control are needed, if
a control is desired ,uterus, small a control is desired ,uterus, small
intestine, appendix or fallopian tube will intestine, appendix or fallopian tube will
provide good material.provide good material.
Wear gloves, lab coat. avoid contact and Wear gloves, lab coat. avoid contact and
inhalationinhalation

17. ResultsResults
Nuclei…………………………….black, blueNuclei…………………………….black, blue
Muscle.cytoplasm,fibrin………..redMuscle.cytoplasm,fibrin………..red
Collagen………………………...greenCollagen………………………...green

19. Nuclei red, collagen green, Nuclei red, collagen green,
cytoplasm redcytoplasm red

20. Infracted rat heart showing Infracted rat heart showing
damaged cardiac tissue in blue.damaged cardiac tissue in blue.

21. Terminal ileum with increase in subepithelial Terminal ileum with increase in subepithelial
collagen table in a patient with collagenous collagen table in a patient with collagenous
colitiscolitis