TEM of cells and tissues

Summary

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.

Fixation

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.

Example Method

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. 

 

 

Nature Neuroscience cover

Brain tissue of a Rat, prepared using TEM techniques.