Improved procedures for pineal gland fixation for electron microscopy

The common procedures used for preparing some organs and tissues for electron microscopy, in which a fixative with the buffer portion adjusted to near-isotonicity to plasma is perfused in vivo, causes intolerable shrinkage of rat pineal cells. The present study was undertaken to optimize the parameters involved in the fixation of the pineal gland. The buffer and its concentration and the aldehyde or aldehydes used were among the variables investigated. The buffers tried were phosphate, cacodylate, PIPES, and HEPES. Decreasing the buffer concentration prevented shrinkage with all four buffers. The optimum concentrations were 0.05 M phosphate, 0.07 M cacodylate, 0.05 M or 0.057 M PIPES, and 0.1 M HEPES. PIPES and HEPES were clearly superior in retaining cytoplasmic density when compared with phosphate or cacodylate. The use of lithium PIPES and HEPES instead of the sodium equivalents enhanced membrane detail. A small volume of more concentrated aldehyde fixative perfused ahead of the main perfusate (a strong prewash) definitely helped prevent shrinkage. Using a mixture of aldehydes consisting of glutaraldehyde, formaldehyde, and acrolein reduced the tendency for shrinkage when compared with glutaraldehyde only. Some of the shrinkage space artefacts could be easily misinterpreted as normal features. Since the pineal gland commonly contains degenerating structures, a dependable fixation procedure is particularly needed. Also, accurate preservation is essential in the evaluation of physiological changes.