Biological specimen Preparation
Specimen preparation can be provided as a service or users can be trained to use equipment for subsequent self-access.
TEM Preparation includes:
Negative staining
We can provide Formvar film grids, Formvar/carbon grids, continuous carbon films, holey carbon films or lacy carbon films as appropriate. Grids can be glow discharged in the Edwards 306 turbo to make them hydrophilic. Nickel supports can be provided for immuno-gold negative staining. stains available are uranyl acetate, PTA or ammonium molybdate.
Negatively stained HPV virus with immunogold label to surface protein
(courtesy of Dr Wei Zhang)
Fixation and thin sectioning for ultrastructure
We can provide , acrolein, glutaraldehyde and formaldehyde based fixatives. Cells and tissues can be embedded as a service or users can to taught to process their own samples and be charged for consumables and instrument access only. We routinely embed in Quetol 651 epoxy resin. Two Leica Ultracut E's are available for self access.
Fixation and thin sectioning for immuno-gold labelling
Various methods are available for post embedding immuno-gold labelling. We can attempt antigen retrieval from archive material fixed in glutaraldehyde and osmium and embedded in epoxy resin (rarely successful). Alternatively we can advise how to test new antibodies for sensitivity to fixation using secondary antibody fluorescence methods. We can then advice if there is a method that may be suitable after this preliminary screening. We have equipment for cryo-immobilisation (Leica CPC) and 2 Leica AFS freeze substitution and low temperature embedding units. Tissues that are coloured or pigment are not suitable for low temperature embedding with UV polymerisation as the colour adsorbs the UV. In that case we can use either LR White or gold as embedding media (contact jeremy for advice).
Slam frozen bacteria freeze substituted and embedded in epoxy resin.
Proteous mirabilis frozen and embedded in HM20 and immonolabelled for the curved norphplogy protein (cuortsey of Dr Daniel Gigyi)
SEM Preparation includes:
Samples for examination in the FEGSEM must either be dehydrated fully or frozen and viewed using the cryo-transfer stage. Two methods of drying are available.
Air drying
Very robust samples (bone, glass or mineral) can be dried by allowing their water to evaporate with gentle heating. Biological sample collapse to < 20% of their original volume and there is gross distortion of their surfaces. this is due to the surface tension changes ant the water/gar interface.
Critical point drying
Samples are dehydrated in organic solvents and transferred to the Polaron CPD where the solvent is replaced with liquid CO2. The unit is sealed and warmed to 37 degrees C where the liquid CO2 changes state to gas. This is vented and eliminates the surface tension changes seen in air drying from water or solvent. This is a pressure system and as such is operated by staff members only.
Freeze drying
This can be carried out in the Edwards 306 but as a service only.
Conductive metal coating
Sputter coating with 1n or more of Au or Cr can be carried out in the Quorum K756X sputter coater. Carbon can be evaporated on to samples with the Edwards 306 for EDX of freeze dried cells or sections. Histology Wax and methacrylate resin embedding and sectioning This is available as a service or for self access to two microtomes. Leica VT 1000 vibrating blade microtome This is available for self access to slice fixed or fresh tissues. fixed tissues can be cut to 20µm and fresh tissue to 150µm. Leica CM 1000 cryostat This is available as a service or self access for cutting frozen sections of fresh or fixed tissues. Self access users are recommended to buy their own knife and guide plate.
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