immersion fixation

Immersion fixation is one of chemical fixation methods for scanning electron microscope (SEM) observation of biological specimens. The method is most widely used for chemical fixation.
In the method, the specimens such as cells and tissues extracted from living organisms are directly immersed into a fixation solution, and proteins or phospholipids in the specimens are fixed.
First, to fix proteins, the specimens are immersed in a paraformaldehyde solution (concentration: about 4.0%) or a glutalaldehyde solution (concentration: about 1.0 to 2.5%) for one hour or longer (pre-fixation). Since paraformaldehyde has high penetration rate into the specimen and glutalaldehyde has high fixation strength, a mixed solution of both chemicals is used in many cases. (For tissues extracted from living organisms, they are cut into small pieces (5 mm square or less) to improve the penetration of the fixation solution. After washing them in a buffer solution, they are rapidly immersed in the fixation solution.) Next, to fix phospholipids of biological membranes, the specimens are immersed in an osmium tetroxide solution (concentration: about 1.0%) for approximately one hour (post-fixation).
It should be noted that the fixation of the proteins by aldehyde (high penetration rate) is carried out in advance of the fixation of the phospholipids because osmium tetroxide has low penetration rate into the specimen and can destroy the proteins.