Cross-linking with formaldehyde

The most common fixative for fluorescence microscopy is formaldehyde. Formaldehyde does not cross-link as effectively as glutaraldehyde, and is therefore not particularly suitable by itself for samples for electron microscopy. The advantage of formaldehyde is its low molecular weight, which allows it to penetrate well into biological samples, including thicker specimen such as tissue slices and whole organisms. Because formaldehyde has only one reactive group, the autofluorescence casued by unreacted aldehyde groups is significantly lower compared to glutaraldehyde. Formaldehyde is commercially available as stabilised formalin solution and can be conveniently used diluting it to the required concentration. However, added stabilisers such as methanol at relatively high concentrations should be taken into consideration, because methanol might cause shrinkage of up to 50% of the sample. Thus, the preferred method is to freshly prepare a formaldehyde solution buffered in PBS at final concentrations of 1-8%, using crystalline paraformaldehyde . It should also be taken into consideration that the cross-linking with formaldehyde is pH-sensitive.

Cross-linking with glutaraldehyde

Alternatively, glutaraldehyde can be used for fixation. Since it has two reactive aldehyde groups it is extremely efficient in the preservation of cell structures and thus preferred in light and electron microscopic approaches in order to study the fine morphology of cells. The disadvantage is the higher molecular weight, which results in a limited penetration and thus it is not particularly suitable for fixation of thick tissue slices and whole organsims because it only preserves the out layers of the specimen. If the second aldehyde group does not react, it causes a siginificant degree of backgound fluorescence. This can be avoided by using low concentrations (≤ 2%) and by treating the sample with reducing reagents such as sodium borohydride or amine-containing reagents such as ammonium chloride. Glutaraldehyde can also potentially mask amine-containing epitopes, which should be taken into account. Glutaraldehyde or a mixture of form- and glutaraldehyde are the preferred method of fixation for correlative microscopy. Due to the more effective cross-linking of glutaraldehyde, it might decrease antibody penetration into cells and tissues.

Cryofixation

This method employs quick freezing of a sample by cutting tissue sections with a vibratome on a cooled block or plunging samples into liquid nitrogen. It presumably is the best way to preserve ultra structures of cells and avoids the introduction of artifacts caused by fixatives or relatively slow fixation with aldehydes, although it is not very suitable for processing adhering cells.

Blocking

Most cross-linking reagents for fixation, such as form- or glutaraldehyde, contain highly reactive aldehyde groups that need to be blocked to avoid unspecific cross-linking in the following labelling steps. Using either reducing (e.g. NaBH4) or amine-containing reagents (e.g. NH4Cl) to convert free aldehyde groups.

Permeabilisation

To prepare the specimen for subsequent labelling, the cells and their strucutres need to be permeabilised by removing the lipids. This is usually carried out by incubating in mild detergent-containing solution (e.g. 0.1-1% Triton X-100, Tween 20 or NP-40.

For more detailed information or protocols and recipes of fixatives see the corresponding webpages at IHC World. Although the protocols are increasingly hidden between commercial information and advertisements on this website, the protocols are there and are usually very helpful.