MASSONS MASSONS TRICHROME STAINTRICHROME STAIN

PRESENTED BY:PRESENTED BY: MEHTAB SHAH MEHTAB SHAH B.S MT 3B.S MT 3RdRd

year year (SDLS(LNH)(SDLS(LNH)

SPECIAL STAININGSPECIAL STAINING

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

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.

MuscleMuscle

Muscle cells are specialized for Muscle cells are specialized for contractility, and they can be classified contractility, and they can be classified according to their microscopic appearance according to their microscopic appearance into 3 categories: into 3 categories:

Smooth muscleSmooth muscle which is found primarily which is found primarily in tubular organs. in tubular organs.

Skeletal muscleSkeletal muscle which is generally which is generally attached to bones. attached to bones.

Cardiac muscleCardiac muscle which is found in the wall which is found in the wall of the heart. of the heart.

SMOOTH MUSCLESMOOTH MUSCLE(involuntary)(involuntary)

Is developed from mesenchymal cells.Is developed from mesenchymal cells. cell is spindle-shaped with an elongated cell is spindle-shaped with an elongated

centrally located nucleus.centrally located nucleus. Microscopically; cells appear eosinophillic Microscopically; cells appear eosinophillic

the presence of glycogen.the presence of glycogen. The cytoplasm contains bundles of The cytoplasm contains bundles of

myofilaments called myofibrils.myofilaments 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 chromatin pattern stain with hemotoxylin and have elongated appearance: they may and have elongated appearance: they may contain one or two nucleoli.contain one or two nucleoli.

COLLAGENCOLLAGEN

These connective tissue protein provide These connective tissue protein provide tensile strength.tensile 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.

ContdContd……………..……………..

Other collagen may form extra Other collagen may form extra cellular matrix meshwork, participate cellular matrix meshwork, participate in cellular anchorage.in cellular anchorage.

PRINCIPLEPRINCIPLE

Trichrome procedure are three dyesTrichrome procedure are three dyes Sections are first stained with an acid Sections are first stained with an acid

dye such as Biebrich scarlet.dye such as Biebrich scarlet. All acidophilic tissue elements such All acidophilic tissue elements such

as cytoplasm. muscle and collagen as cytoplasm. muscle and collagen will bind with the acid dyes.will bind with the acid dyes.

The section are then treated with The section are then treated with phosphotungstic and phosphotungstic and phosphomolydic acid.phosphomolydic acid.

Contd…………………….Contd…………………….

Because cytoplasm is much less Because cytoplasm is much less permeable than collagen the permeable than collagen the phosphotigstic and phophomolydic acids phosphotigstic and phophomolydic acids cause the Biebrich scarlet to diffuse out of 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 Finally a dye of large particle size such as light green is used that is able to enter light green is used that is able to enter collagen.collagen.

FIXATIVEFIXATIVE Bouin’s solution is preferredBouin’s solution is preferred10% neutral buffered formalin 10% neutral buffered formalin

may be usedmay be used

TECHNIQUETECHNIQUECut paraffin section at 4 micronsCut paraffin section at 4 microns

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

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;

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 Phosphotungstic – phosphomolydic acid solutionsolution

10-15 mint;10-15 mint;

Light green solutionLight green solution2-5mint;2-5mint;

Dehydrate in absolute alcoholDehydrate in absolute alcohol

Clear in xyleneClear in xylene

Mount in synthetic resinMount in synthetic resin

MicroscopyMicroscopy

Quality control and safetyQuality control and safety

Practically every tissue has an Practically every tissue has an internal control so no other control internal control so no other control are needed, if a control is are needed, if a control is desired ,uterus, small intestine, desired ,uterus, small intestine, appendix or fallopian tube will appendix or fallopian tube will provide good material.provide good material.

Wear gloves, lab coat. avoid contact Wear gloves, lab coat. avoid contact and inhalationand inhalation

ResultsResults

Nuclei…………………………….black, Nuclei…………………………….black, blueblue

Muscle.cytoplasm,fibrin………..redMuscle.cytoplasm,fibrin………..red

Collagen………………………...greenCollagen………………………...green

Nuclei red, collagen green, Nuclei red, collagen green, cytoplasm redcytoplasm red

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

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