Transmission Electron Micrsocopy (TEM) imaging of cells and tissues generally requires a number of preparation steps. These include a fixation step, which is either cryo fixation, or chemical fixation, followed then by staining, and embedding phases.
This part of the preparation requires the material to be immobilized as rapidly as possible so that the biological processes that are of interest can be observed in their most native state. This is done either by chemical fixation or rapid freezing.
The method outlined was used for the proccessing of brain tissue;
– Perfusion fix tissue, using 2.5% glutaraldehyde abnd 2% paraformaldehyde in 0.1M PB pH 7.4
– Vibrotome, cutting brain into 80 micron thick sections
– Postfix, in 1% osmium tetroixde and 1.5% potassium ferrocyanide
– Stain, in 1% uranyl acetate.
– Dehydrate, using graded alcohol series
– Infiltrate with resin (Durcapan)
– Embed between coated glass slides.
– Polymerise, at 65 degrees C overnight.