staining mechanisms(8/12)

staining mechanisms(8/12)

1. 3. metallic impregnation

   • 1.deposition of metallic silver onto tissue elements
   • mechanism
     • argyrophil reaction
     • e.g.capable of impregnation with silver salt with the use of an external reducing agent(e.g. stain reticulin)
     • require molecules
       • formaldehyde or hydroquinone
     • *no external reducing agent
       • argentaffin:tissue contains reducing groups to reduce silver ion, without the need for external reducing agent
       • example of argentaffin 1.enterochromaffin cells(is a type of enteroendocrine cells-know as kulchitsky cells) 2.melanin

2. 2 elective solubility

   • criteria of a lysochrome(a soluble dye used for histochemical staining of lipid)
   • features
     • 1.strong coloured 2.highly soluble in tissue constitutes than in solvents(e.g. soluble in tissue lipid) 3.solvent will not dissolve specimen
   • example dye and solvent
     • 1.oil red O 2.sudan (III,IV and black) 3. nile blue sulphate(NBS)
     • Solvents: 1.70% alcohol 2.60% triethyl phosphate 3.propylene glycol 4.60% isopropanol

3. 1.vital stain*

   • properties
     • 1. non toxin, harmless stain: colored tissues or cells of living organisms. 2. often used to determine live/dead cell ratio in a cell population
   • types
     • supravital
       • it is processed by removing living tissues from body and cells and treated with a non toxic dye solution
     • intravital
       • a stain absorbed by living cells after injection into the body

4. 4.histochemical reactions

   • 1.It may utilize a dye or in itself to produce coloured reaction e.g.. a. Schiff reagent uses basic fuchsin: coloured aldehyde site magenta
   • 2.enzyme histochemistry

5. 5.staining with dye

   • based on different kind of chemical bondings
   • 1.ionic bonds
     • it is electrostatic interaction between oppositely charged ions 
     • enhancement
       • 1.binding of dyes depend on tissue ionization 2.pH(can get highly specific staining): -acid pH favours staining with anionic dyes -alkaline pH favours staining with basic dyes
     • factors can affect the staining
       • how salt/acid concentration affect tissue ionization and staining??
       • acid level different
         • concerning tissue components
         • stronger
           • phosphate groups in nucleic acids and sulphate groups found in mucin:can still ioniz and less easily inhibited
         • slightly acid solution
           • e.g. methylene blue will act as a differential staining:stain nuclear but unstain proteins
       • salt concentration
         • in high salt concentration:competition between dyes and the salt ions, so staining will be inhibited  -weak salt can increase the staining.
       • pH
         • alcian blue staining mucin
           • apparent staining is produced in ideal pH environment: alcian blue can bind to both weak carboxylic-acid containing mucin and strong sulphated mucin.
   • 2.hydrogen bonds
     • an intermolecular force:between a hydrogen atom in one molecule and a small atom of high electronegativity in other molecules.-play a role in the selectivity of the dye -not affected by pH and salt concentration ,but stronger of the hydrogen-bonding agents.
     • Application
       • 1.Congo red for amyloid 2.Best carmine's for glycogen
     • MOA
       • used to inhibit ionic bonds, increase the selectivity of the dye
       • 1.use non-aqueous solvent
         • water inhibit hydrogen bonding and favours ionization
       • 3.extreme pH
         • chosen to inhibit the ionization of tissue groups
       • 2.high salt concentration
         • competition with the dye
   • 3.van der waals force
     • attraction between non-polar molecules: weak, short range forces
     • e.g. orcein for elastin
   • 4.covalent bonds
     • very strong bonds and are not easily broken: important in PAS(periodic Schiff reagent: histochemical technique)
   • 5.hydrophobic interactions
     • -not chemical bond -important in selectivity and play a role in lipid staining

6. selective techniques

   • stain several structure: differentiate by other features

7. specific techniques

   • only a particular tissue structure or chemical moiety is demonstrated

8. Important staining***

   • a.Haematoxylin
     • -natural product,no obvious chromophore -for staining: first oxidized to heamatein
     • features
       • 1.oxidation is slower in acid conditions: many solutions kept acidic: (i)ripening-oxidation of heamatoxylin to haematein in atmospheric oxygen. (ii)using oxidizing agents-sodium iodate, potassium permanganate or mercuric oxide
     • mordants for haematoxylin
       • Aluminium: usually potash alum and ammonium alum
       • aluminium
         • solubility different in pH: 1.in acid solution: the alum dye lakes are quite soluble and have a strong red color 2.in alkaline solution: dye lakes are less soluble and have a strong blue colour.
       • iron-Hx
         • -salts used are ferric chloride or ferric ammonium sulphate(ferric salt)-blacker and intense .will resist acidic counter stain -ferric salts are oxidizing agents and will accelerate oxidation of haematoxylin to haematein
         • -mordant is therefore either used separately or -mix mordant and haematoxylin before use
   • b.Masson-Fontana silver method
     • 1. required argentaffin: substances that reduce silver diamine e.g. for melanin demonstration/5 hydroxytrypatmine in enterochromaffin cells
     • process for the demonstration: 1.blenching of melanin on one slide  MOA:Ag impregnation reducing groups convert silver diamine to colloidal metallic silver
   • c.Jone's Methenamine Silver
     • lt is used to stain glomerular basement membrane MOA:argentaffin metallic impregnantion
     • The structure can be demonstrated: 1. spikes of GBMs due to immune deposits in membranous glomerulonephritis 2.spicules in amyloidosis:due to amyloid infiltration of peripheral capillary loops  3.double contours: mesangial interposition in membranoproliferative glomerulonephritis.
     • staining mechanism
       • 1. mild oxidation: expose reactive aldehyde groups 2.Ag impregnation: Ag+ bind to aldehyde groups and reduced to Ag deposit

9. reticular fibers(the general demonstration)***

   • -agyrophilic -common in the liver,kidney and spleen(perform a support function) -demonstration by silver stains
   • 1.sensitization
     • metallic complexes is developed at the aldehyde groups
   • 2.silver impregnation
     • 1.silver salt and ferric complex exchange ions 2.ammoniical silver solution is reduced by the exposed aldehyde groups to metallic silver
   • 3.developing
     • 1.further reduction of the diamine silver by transfer the section to formaldehyde