Enhancements, Chapter 13
Enhancement–13.1 A generic fixation procedure.
As a starting point for a 1mm3 cube of tissue or a 0.5mm thick section, use as a primary fixative, a solution of 0.5 to 4% glutaraldehyde and 0.5 to 2% formaldehyde for up to 30min at room temperature. A suitable buffer for plant and microbial material would be 100mM cacodylate or phosphate solution, pH 6.8 to 7.2. For animal material, 120mM phosphate buffered saline, pH 7.2 to 7.4, is a good starting point. Choose a buffer which has a pKa within 0.5 units of the desired pH. As a general rule, the volume of the fixative solution should x100 greater than the volume of the specimens. This same ratio should be maintained during dehydration and resin infiltration. If the tissue samples float on the surface of the fixative, the container should be placed into a vacuum desiccator and subjected to sufficient vacuum pressure to cause bubbles to appear in the fixative solution. Allow the container to return to atmospheric pressure and repeat the process until the specimens sink. Do not process floating specimens.
After the primary fixation is completed, the sample should be washed several times over a period of 1hour in the same buffer used as the fixative vehicle and place in unbuffered 1% osmium tetroxide for no more than 2 hours at room temperature. The samples should be gently agitated during the fixation procedure. The osmium solution acts as a secondary fixative. After fixation the sample should be washed several times in distilled water to remove traces of fixative and buffer salts.
Fixation times vary enormously; plant tissues generally need longer fixation times than animal and microbial specimens. Larger and impervious samples will need longer fixation times. Much care must be taken with all the components of fixative vehicle that may need to contain additives to enhance the preservation of particular structures. Non-wettable samples can be pre-treated with gamma glycidoxypropyl trimethoxysilane. Vapour fixation can be used for very delicate small samples and for aerial structures.
Enhancement-13.2 A generic procedure for conductive staining
A typical mixture would be to first fix the sample in an aldehyde fixative followed by 2% osmium tetroxide and then to immerse it into a 3% solution of thiocarbohydrazide. The thiocarbohydrazide cross links to the osmium and retains active sites to which additional osmium may be attached. The osmium-thicarbohydrazide cycle is repeated several times to load the sample with heavy metal. Other mixtures include tannic acid and osmium, hydrazine and osmium vapour and silver nitrate alone. In addition to increasing the bulk conductivity of the sample the process also toughens the sample.
Enhancement-13.3. A generic physico-chemical dehydration procedure.
A 1mm3 fixedspecimen is placed first in 10ml of a 30% aqueous solution of ethanol and after 20min half the dehydrating fluid is removed and gradually replaced with an equal volume of a 50% solution. This process is repeated every 20min with 75, 85, 95 and 100% ethanol and finally through two or changes of 100% ethanol dried over molecular sieve (Linde Molecular Sieve 3a). Caution, do not shake the bottle and only remove the ethanol using a pipet to avoid contamination with fine particles of drying agent. In the final stages of dehydration it is important to keep air tight lids on the containers to prevent atmospheric water vapour being absorbed by the ethanol The dehydration process takes about 3 hours and should be continuous, but if necessary, the sample can be left in the 75% ethanol for up to twelve hours. The samples should be gently agitated during the dehydration process.
Enhancement-13.4 A generic procedure for critical point drying.
The process is carried out as follows bearing in mind any caveats and recommendation s suggested by the equipment manufacturer. The apparatus should be pre-cooled to 10-12oC with the lid firmly closed. The samples must remain immersed in either dry acetone or ethanol at all times and as they are quickly transferred to the apparatus. The lid should be sealed tightly and the appropriate valves opened or closed to allow the specimens to be thoroughly flushed several times with liquid CO2 at a pressure of 1200psi. Make sure the liquid CO2 comes from a clean container. The flushing is necessary as the two dehydrating fluids are not completely miscible with liquid CO2. The specimens must remain under liquid throughout the flushing and substitution. Once the substitution is completed, the appropriate valves are opened and closed and the temperature raised to 32-34oC. The pressure rises to about 1500psi and the liquid CO2 first reaches and then passes the critical point and is converted to the gas phase without surface tension effects. The temperature should be kept at 32-34oC and the appropriate valves opened and closed in order to very slowly reduce the internal pressure. Once atmospheric pressure is reached, the door is opened and the now bone dry samples quickly transferred to a sealed container which contains a good desiccant such as Linde Molecular Sieve 3A.
Enhancement-13.5. A generic procedure for rapid epoxy resin embedding.
This normally takes about 1-2 days but it is possible to accelerate the process to about 15 hours. Fixed and dehydrated 1mm2samples, immersed in 100% dry ethanol, are substituted for 10min in a 1:1 mixture of ethanol and propylene oxide and then passed to 100% propylene oxide for a further 10min. The sample is then infiltrated in 1:1 Spurrs low viscosity resin : propylene oxide for 60min, 100% resin overnight , passed through another change of pure resin for a further 2hours before being put into moulds with fresh resin and catalyst and cured at 60oC for 16 to 24 hours. The specimens should be gently agitated during the initial stages of resin infiltration. The book by Glauert and Lewis (2000) has a good collection of resin recipes and embedding procedures.
Enhancement-13.6. A generic wet chemical fixation routine for immunocytochemical studies.
Mild fixation in 2% formaldehyde + 0.05% glutaraldehyde in either a phosphate or PIPES buffer, followed by progressive lowering of temperature dehydration to 80% ethanol and slow infiltration in a hydrophilic acrylate resin. The polymerised resin is then sectioned or fractured to reveal the specimen. Applying microwaves during fixation gives an enhanced expression of the antigens. The antigenic sites may be localised by either the direct or indirect method discussed earlier. Much experimentation may be needed to ensure there is a high specific signal and a low non-specific background.
Chapter 13, Enhancements FINAL111/16/18