Fixation for IF/ICC
The first step in an IF/ICC procedure is fixation. This prevents the cells from decomposing and maintains the subcellular structure as well as the cellular proteins. There are a variety of fixatives that can be used, so to find the best method multiple fixatives should be tested. Fixation methods are roughly divided into two groups: chemical crosslinkers and organic solvents. Chemical crosslinkers form intermolecular bridges through free amino acids, whereas organic solvents work by removing lipids and denaturing the proteins. Fixation in cells is usually achieved by removing the culture medium and replacing with the fixative solution, but this can damage some cell types and in this case a fixative in a higher concentration can be added to the culture solution.
Formaldehyde is the most common fixative used, it is dependent on the methylene bridges that are formed between proteins and nucleic acids. It does not, however, crosslink lipids and produce vesiculation of the plasma membrane. An advantage of using formaldehyde as a fixative is that it preserves cellular morphology well due to the crosslinks created. Overfixation with formaldehyde can cause antigens to become crosslinked as well which reduces the antigenicity, but by adding an antigen retrieval step it can unmask the antigens.
Glutaraldehyde is a dialdehyde and has two reactive groups for fixation. It is more rapid at fixing than formaldehyde and is more effective at forming crosslinks, however this can be a disadvantage as it may destroy antibody binding sites. Glutaraldehyde also produces high auto fluorescence.
EGS (ethylene glycol-bis-succinimidyl succinate)
EGS also works by crosslinking primary amino groups, and allows for intracellular crosslinking as it is membrane permeable. The major advantage of using EGS is that the crosslinks are reversible which may be used to restore antigen binding sites. EGS is useful for intracellular and membrane bound proteins.
The most common alcohols used as fixatives are methanol and ethanol. These fix the cells by rapidly dehydrating and disrupting hydrophobic and hydrogen bonding, exposing the internal proteins. An advantage of this method is that it is a faster procedure compared to chemical crosslinkers and also has good preservation of the cellular architecture. However it is perceived that alcohols are not as efficient at preserving tissue morphology as aldehyde based fixatives. Alcohols can provide high levels of immunostaining as well as low levels of non-specific staining.
Acetone is also a coagulating fixative, which changes the hydration state of the cellular components. It is also a faster procedure as it does not need a permeabilisation step. It can sometimes be less damaging to epitopes than alcohols, leading to a better histological preservation of the cell. However, like alcohols, soluble compounds and lipid components can be lost.
|Chemical crosslinkers||Formaldehyde||Crosslink proteins through free amino goups||preserves cellular morphology and is good for already fluorescent proteins||Antigens might be crosslinked|
|Glutaraldehyde||Preserves cellular morphology and is good for fluorescent proteins||Antigens might be crosslinked and gives off a high level of fluorescence|
|EGS||The cross links that are formed are reversible|
|Organic solvents||Methanol||Dehydrogenation and protein precipitation.||Cellular architecture is well preserved and is a faster procedure.||Has a strong negative effect on epitopes, is not appropriate for fluorescent proteins. Soluble compounds and lipid components are lost.|
|Acetone||Epitopes are greater preserved and is a faster procedure||Not appropriate for fluorescent proteins. Soluble compounds and lipids components are lost|