Molecular and cellular analysis Flashcards
Histology
Process of staining
Fixation -> graded alcohols 70% alc -> Xylene -> Paraffin wax -> microtomy -> xylene -> graded alcohols 70% -> water -> staining -> dehydrate in alcohol -> clear in xylene -> mount in DPX
IHC
Immunohystochemistry is critical in determining the exact subtype of cancer/ grade.
FIXATION
- Maintain tissue morphology and stabilise proteins.
- Fixatives crosslink proteins
- Soluble proteins are fixed to structural proteins and rendered insoluble providing strength.
- Inhibits bacterial/fungal growth.
CONS FIXATION
- Damages proteins
- Has to be carefully optimised for enzyme histochemistry, immunocytochemistry and electron microscopy to preserve antigen sites.
- Balance: retain biological activity vs good morphology.
FIXATION
It is important that fixatives are ISOTONIC.
Isotonic - Any external solution that has the same solute concentration and water concentration compared to body fluids.
Hypertonic (cells lose fluid and shrink)
Hypotonic (cells swell and rupture)
ALDEHYDES
- Formaldehyde, glutaraldehyde - most used in histopathology.
- Formaldehyde in solution is called formalin.
- Cross links are formed within and between protein molecules (especially amino acid lysine).
- Aldehyde reactions are pH dependant faster at higher pH.
- Formaldehyde is reversible with water within 24 hours.
- Glutaraldehyde is rapid and irreversible. (many cross links).
CONS ALDEHYDES
- Glutaraldehyde can cause up to 30% of the alpha helical structure of protein.
- Protein alterations lead to changes in isoelectric points and staining properties in the tissue.
- TOXIC
- Slow (over night).
Types of embedding
Paraffin wax - Light microscopy is the main method. cheap simple and easy to automate.
Frozen sections - used to demonstrate sub cellular locations of enzymes using immunofluorenscence. lipid analysis.
