Chemical fixation
A number of factors affect the quality of fixation including pH, temperature, osmolarity, fixation time, and sample size, these are likely to have to be optimised for your sample.
The most commonly used fixation protocol for resin embedded samples involves primary fixation with glutaraldehyde (1-2%) followed by secondary fixation with OsO 4 (2-4%).
For immuno-studies, paraformaldehyde (4-8%) is often preferred as the primary fixative, sometimes with low concentrations of glutaraldehyde (0.1-0.5%), before cryo-preservation because many immuno-reactions are glutaraldehyde sensitive. For studying soluble proteins, strong (2%) glutaraldehyde fixation is needed to reduce protein extraction. Alternatively, immuno-staining of membrane and cytoskeletal proteins may be enhanced by extraction of cytosolic proteins.
Properties of common fixatives:
Handling of fixatives should only be performed in a fume cupboard, stock solutions should be stored in fridges or freezers where no unfixed biological reagents are kept (such as antibodies and enzymes) to prevent loss of their activity.
Paraformaldehyde
- Formaldehye reacts with many functional groups on proteins including amine, thiol, hydroxyl, imidazoyl and phenolic groups
- The majority of reactions are reversible and so extensive wash steps should be avoided
- Formaldehyde does not cross-link lipids but unlike glutaraldehdye, can cross-link DNA.
Glutaraldehyde
- The best fixative for preserving fine structure, cross-links proteins rapidly and irreversibly
- Targets protein amino groups
- Lysine is the most important component of proteins although glutaraldehdye also reacts with other amino acids including cysteine, histidine, tyrosine and tryptophan
- It probably also reacts with free amino groups on some lipids (eg. PS and PE) although may not prevent lipid extraction during subsequent processing for resin embedding unless secondary OsO4 fixation is also performed
- Stock (25%) glutaraldehyde solution is relatively stable at 4°C or -20°C however, shelf-life is limited to a few weeks once added to a buffer.
Osmium tetroxide
- Most important features are its ability to cross-link lipids and the electron density of reduced osmium that can provide a scaffold for further staining by lead to increase contrast
- Primarily used as a secondary fixative because of its slow tissue penetration rate and poor ability to cross-link proteins and carbohydrates
- The most likely lipid targets are unsaturated fatty acids
- OsO 4 reacts with certain protein side chains including thiol, hydoxyl, phenolic, carboxyl and amino groups; cyteine and methioine appear to be the most reactive amino acids towards osmium tetroxide.
Rate of infiltration of samples
The penetration rates of fixatives are limited but can be improved with co-incubation, for example:
- 2% glutaraldehyde fixation for 2 hours can produce good fixation up to a depth of 1mm
- This may be increased to as much as 5mm if 2% paraformaldehyde is also included.
This is related to the fact that paraformaldehyde penetrates tissue much more rapidly, increasing permeability of the sample for more effective glutaradehyde penetration. In practice, preparing sample blocks of less than 1mm3 are the best way to maximise the chances of good fixation.
Cryo-fixation
The main drawback of chemical fixation is that it is relatively slow allowing physiological changes to occur before fixation is complete or preventing rapid events from being imaged effectively. Chemical fixation can also induce shrinkage or swelling and changes to organelle morphology for example aldehyde fixative causes a significant reduction in endosomal volumes.
Cryo-fixation avoids these problems through ultrarapid freezing of samples preserving them in as near a native state as possible. To achieve this, the water in the sample must form amorphous rather than crystalline ice which tears the sample apart. Crystalline ice forms at temperatures down to -143°C therefore, in order to generate amorphous ice the sample must reach temperatures below this almost instantly. This means that only thin films (e.g. of virus or protein suspensions) can be prepared as frozen hydrated specimens unless more sophisticated techniques such as high pressure freezing are used. High pressure freezing lowers the vitrification temperature to approximately -100°C, increasing the depth of efficient amorphous ice formation from 10-20µm associated with plunge and slam freezing to 100-200µm.
An alternative approach for large tissue samples involves cryo-protecting the specimen with protectants such as sucrose, glycerol or PVP, often after pre-fixation with aldehydes to facilitate access of cryoprotectants. The samples are plunge frozen in liquid nitrogen and sectioned on a cryo-ultramicrotome for subsequent immuno-labelling and/or morphological analysis. Although this procedure can involve chemical pre-fixation, it avoids some of the major problems of extraction associated with dehydration during processing for resin embedding, preserving much more of the native state and antigenicity within the sample
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