Chemical fixation is a common technique used to preserve cellular and tissue structure for biological samples. Ideal fixation processes should happen quickly to minimize artifacts. The fixatives enter the cells by diffusion. To minimize artifacts, consideration should be taken on the size of the sample (the smaller the better), pH, osmolarity and temperature of the buffer, fixation chemical, and fixation time.
Choice of Chemicals:
Aldehydes such as glutaraldehyde and formaldehyde are the most commonly used fixatives.
Glutaraldehyde is great for crosslinking proteins while formaldehyde, due to its small molecular size, can penetrate into cells quickly; however, aldehydes lack the ability to fix lipids present in the sample.
(2) Osmium Tetroxide
Osmium tetroxide is excellent for fixing and preserving membrane structure for TEM observation and adds electron density to the sample.
(3) Acetic Acid
Acetic acid is often used to quickly fix samples; however, because the cells are dehydrated quickly, cell shrinkage is a common artifact. This is often used to observe tissue samples such as leaf vein patterns.
25% Glutaraldehyde EM grade (ampoule) in the fridge
16% Formaldehyde EM grade (ampoule) in the fridge
2% Aqueous Osmium solution in the fridge
0.2M Sodium cacodylate buffer or 0.2M Phosphate saline buffer
The fixative is a mixture of buffered chemicals that is made in the fumehood just prior to fixation due to the chemicals toxicity.
For example, commonly used aldehyde fixatives in the BIF are 2.5 % Glutaraldehyde and 4 % formaldehyde in 0.1M Sodium cacodylate buffer.
Depending on the sample being fixed, the choice of fixative will vary.
The samples need to be completely submerged in the fixatives to ensure the cells are properly preserved.
For the sample having thick cuticles and cell walls, fixation can be done under vacuum to facilitate the diffusion of chemicals into the cells.