Scanning Microscopy


Many techniques for processing tissue into resin are available, varying from conventional room temperature to low temperature procedures. The problem is to choose an appropriate method to suit the biological specimen under study. Room temperature approaches with aldehyde and osmium fixation do not give optimal retention of immunoreactivity. Osmium can be removed from sections, but recovery of immunosensitivity is reduced. Osmium post-fixation can be omitted, but heat polymerization of resins causes tissue extraction and loss of immunoreactivity. Alternative techniques rely on the use of milder polymerization methods and avoid osmium. However, while providing an improvement, this alone is not sufficient to maximize tissue reactivity. Fixation with high concentrations of glutaraldehyde ( > 1%) and processing into resin at either room or low temperature results in retention of similar levels of immunoreactivity. Low concentration glutaraldehyde ( < 0.2 %) fixation for short periods of time ( < 60 minutes) produces improved tissue immunoreactivity and allows low concentrations of antigen at secondary sites to be detected. However, the tissue is now only minimally stabilized and is prone to extraction and conformational damage during processing. It can be partially protected by employing one of two strategies: processing at room temperature with partial dehydration (upto 70% solvent) and rapid embedding in LR White or Lowicryl K4M at 0 °C, or processing at progressively lower temperatures (PLT) and embedding in Lowicryl at -35/-50 °C. In a third strategy, specimens sensitive to very low fixative concentrations are cryoimmobilized, then resin embedded after substitution or freeze-drying (this latter method awaiting evaluation for inclusion in our strategical approach).

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