Sample preparation often involves a trade-off between antigenicity required for immuno-labelling and maximal preservation of ultrastructure.
1. Fixation
The critical first step in sample preparation aims to preserve the specimen as close to its original state as possible. Formaldehyde and glutaraldehyde are the most commonly used primary fixatives and are often used in combination.
Formaldehyde rapidly penetrates tissues cross-linking proteins however glutaraldehyde although slower at penetrating samples is a far better cross-linker. A common mistake is to try to fix too big a sample (i.e. more than 0.5 mm 3) resulting in poor fixation of internal regions and many subsequent problems with sectioning and imaging. Importantly, neither formaldehyde nor glutaradehyde fix lipids, these will be partially extracted unless secondary fixation can be performed with eg. osmium tetroxide which both reacts with lipids and is an electron-dense stain.
Samples processed for resin embedding are subsequently:
2. Stained
Samples are stained with electron-dense stains such as osmium tetroxide, lead citrate and uranyl acetate to maximise contrast for viewing ultrastructure.
3. Dehydrated
Samples are dehydrated through a graded series of alcohols to remove all water.
4. Infiltrated
Samples are infiltrated with increasing concentrations of resin before the resin is finally polymerised in an oven, encasing the sample in a hard plastic suitable for sectioning.
For immuno-labelling and/or maximal preservation of native structure a number of cryo-techniques have been developed.
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