Fixatives commonly used for Electron Microscopy
Glutaraldehyde
Reacts rapidly with proteins and that, being a dialdehyde, stabilises structures by cross-linking before there is any opportunity for extraction by the buffer. Hence more ground substance of the cytoplasm (glycogen) and of the extracellular matrices is preserved. Glutaraldehyde alone is not an adequate fixative, since certain cell components especially lipids, are not fixed and may be extracted during dehydration, therefore secondary fixation is required using osmium tetroxide. Depth of penetration 2 - 3 mm / hour.
Osmium tetroxide
The solution should have a yellow
colour, but may become colourless if stored for too long, if this
occurs discard solution and prepare fresh. Reacts with lipids
and also acts as a stain, osmium will penetrate most tissues at
a rate of about 1mm / hour. Extended times will cause extraction
of many proteins, therefore keep time of immersion to a minimum.
Formaldehyde
Commercially available solutions are unsuitable for electron microscopy because of their content of methanol. Paraformaldehyde powder is used to prepare methanol-free formaldehyde. Main advantage of formaldehyde is that it has a higher rate of penetration than either glutaraldehyde or osmium tetroxide so that large blocks of tissue are well fixed. Will penetrate most tissues at a rate of about 10 mm / hour, but will take a longer time to stabilise the tissue.
Formaldehyde and Glutaraldehyde mixtures
Fixatives containing both paraformaldehyde and glutaraldehyde provides a much better quality of fixation, than either aldehyde alone. Formaldehyde penetrates tissues rapidly and mildly stabilises proteins etc. which are then permanently fixed by the glutaraldehyde.
Uranyl acetate - (EN-BLOC stain)
Used as 2% solution in distilled water , this takes 24 hours to dissolve. The solution will keep for 1 week if stored in dark at 4oC.
Acrolein
This is a highly reactive, volatile and flammable liquid, always handle in fume cupboard. Once opened must be stored in freezer at -15oC. Any spills should be neutralised with 10% sodium or potassium bisulphite solution. Always prepare fixative immediately before use.
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