MTC Flashcards
What is special about masson trichrome
What is the general principle of the stain
only staining technique that has no primary stain (Nuclear stain + 2 counterstains
Relies on the varying densities of tissue
Dyes of varying molecular weight (i.e., size)
What is the purpose of the masson trichrome stain
to be able to differentiate acidophilic tissue components:
RBC, Muscle, Collagen fibers, fibrin
screening tool to see if there are changes in tissue pathology - looks at presence and density of fibrosis
-early vs end stage disease
Classification
Target tissue component:
Staining principle:
Positive controls:
of MTC stain
Classification: Connective Tissue stain
Target tissue component: Acidophilic tissues
Staining principle: Porosity & ionic bonding
Positive controls: Every tissue has an internal control (but may use uterus, small intestine, appendix, fallopian tube)
how is masson trichrome used
two anionic dyes that are negatively charged in solution are applied either together or alone with PMA or PTA
sequential or combination application
how is sequential application done
- small dye molecules ALL go to cationic dye sites
-rinse causing dye to leave sites that are more porous
-PTA and PMA will replace the dye molecules leaving the porous site
-then a larger molecule displaces PTA/PMA
What factors affect trichrome staining
Fixative
pH
Dye concentration
timing in staining reagent
How can fixative affect trichrome staining
NBF makes crosslinkages that masks reactive chemical groups
-need to perform post fixation with Bouins
How can pH affect trichrome staining
lower with acetic acid pH<IEP
-a lower pH (<2.5) makes sure that acidophilic binding sites with a positive charge are accessible to anionic dye
how can Dye concentration affect trichrome staining
-important in dyes that are used in combination
-need a balance or youll have bad staining
how can timing in each staining reagent affect trichome staining
-follow the sops otherwise the stain wont turn out
What is the 1st step in MTC
Fixation
-increase dye binding sites for acid dyes
-tissue needs to be fixed in fixative with mordant properties
Picric acid in Bouin is used for post fixation but note that picric acid with less than 10% water is v expensive
What is step 2 in MTC
nuclear staining
-weigerts iron hematoxylin
-need to use iron hematoxylin because its resistant to effects of acid in counterstaining
-DONT USE HARRIS HX
-attachment with ionic bonding or salt linkage
What is step 3 in MTC
primary counterstain
-dye of small molecular size
-Reagent: Acid fuchsin-Ponceau 2R, Biebrich scarlet-Acid fuchsin (NS)
-Stains all cytoplasmic components like Muscle, RBC’s, collagen
what is step 4 in MTC
differentiator/ trapping agent
-Phosphomolybdic acid/ Phosphotungstic acid (PMA or PTA)
-Decolourizes collagen
-traps the primary stain in muscle, RBC’s and prevents the 2ndary counterstain from staining them
What is step 5 in MTC
-Secondary Counterstain
-the 2ndary counterstain has a dye molecule that is larger than the primary like light green or aniline blue
-stains collagen
What are the results of this stain
-Nuclei – blue-black
-Cytoplasm, muscle, erythrocytes – various shades of red
-Collagen – green (LG) or blue (aniline blue)
