Part of my histology series
Prevention of bacterial growth and autolysis (cells being destroyed by their own enzymes)
Prevent shrinkage, expansion i.e. change of shape during the subsequent processing and various tests
Allow staining of tissues to visualise various tissue components, cells, antigens etc.
Must penetrate evenly and rapidly, and harden tissues
Fixatives have the properties of forming cross links between proteins to form a gel, ideally keeping everything as in vivo
Soluble proteins are fixed to structural proteins & rendered insoluble, & whole structure given mechanical strength
Classifications of fixatives
Aldehydes e.g. formaldehyde, glutaraldehyde (all non-coagulant)
Oxidising agents: osmium tetroxide, potassium dichromate (non –coagulant)
Protein-denaturing (coagulant): acetone, acetic acid, methyl alcohol, ethyl alcohol
Physical: Heat, microwaves
Miscellaneous: Mercuric chloride, picric acid, (coagulants)
Colourless gas, water soluble
In the lab: 10% formal saline/neutral buffered formalin
Acidic groups of the protein are unaffected by formaldehyde, basic groups are blocked.
Does not precipitate DNA in solution.
Most lipids are well preserved, though not necessarily fixed.
Some phospholipids are very slowly dissolved by formalin.
Formaldehyde useful for study of glycogen.
for use with transmission Electron microscopy (TEM).
Fast fixation and penetration of tissue
Primary fixation with glutaraldeyhde; secondary fixation in osmium tetroxide to retain lipids
Occurs as pale yellow crystals
POTASSIUM DICHROMATE (K2 CR2 07 )
Soluble at around 10% in water at room temperature, but insoluble in absolute alcohol.
Proteins converted to gel rather than precipitated.
DNA dissolved; little effect on carbohydrates; useful for lipid demonstration.
Has in the past been used for fixing tumours suspected of being pheochromocytomas.
Protein Denaturing Reagents
ETHYL ALCOHOL, METHYL ALCOHOL, ACETONE
Mainly used for the fixation of cytological material. In some under fixed tissue specimens initially fixed with formalin may undergo some fixation by alcohol during the subsequent ‘paraffin processing’
precipitation, dehydration
Lipids: not preserved/dissolved
Carbohydrates: associated with proteins will also be precipitated
Shrinkage, hardening, morphological considerations
TEMPERATURE FIXATION/STABILISATION
Tissue placed in buffer/saline/isotonic solution
useful for the rapid fixation of routine surgical specimens.
For EM and some histochemistry best done at 0- 4C
Freezing covered in tissue stabilisation post.
MERCURIC CHLORIDE (HG CL2 )
Secondary Fixative reacts and cross links proteins via the sulphydryl (-SH) group of cysteine amino acids
Reputed to give superior morphology for H&E and special stains such as trichrome methods.
Disadvantages; toxicity; mercury pigment; contact with metal; crystalline structure becomes progressively worse in stored blocks; not good for immunocytochemistry & molecular biology.
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