Fixation

If we are to study the location of molecules within cells, then some form of immobilization must take place prior to labeling. Molecules that are not immobilized may be displaced from their normal location resulting in either a false localization or a negative result (if the molecules are washed away, for example).

The easiest method of immobilization is by chemical fixation and aldehydes have been traditionally used for this purpose. The two most popular aldehydes being formaldehyde and glutaraldehyde. For immunocytochemistry tissues or cells can be fixed by immersion in a buffered solution of aldehyde, using the same methods that are used for Epoxy resin embedding. The pieces of material should be small for fast aldehyde penetration but should not be damaged by over manipulation (squeezing, pulling, cutting etc.).

The aldehyde to be used, and the concentration, depends on the antigen to be studied. For example, although glutaraldehyde produces better cellular morphology some antigens are not recognized by antibodies after this fixation. Other antigens seem unaffected even by high concentrations of glutaraldehyde. So called "insensitive" antigens can be fixed in glutaraldehyde concentrations of up to 1% for 15-60 min. For "sensitive" antigens, formaldehyde concentrations of up to 8% can be used (for more details on the process of fixation refer to G. Griffiths 1993 Fine Structure Immunocytochemistry, Springer Verlag, Berlin and Heidelberg).

Whether using formaldehyde, glutaraldehyde or a mixture of the two it is important to note three things:

1. Unlike glutaraldehyde, when used at low concentrations (<4%) formaldehyde cross-linking is partly reversible. It is important to avoid extensive washing after fixation with formaldehyde alone.
2. Again, unlike glutaraldehyde, the cross-linking reactions of formaldehyde occur much slower. For this reason it is best to leave the cells or tissues to be fixed for a longer time than when using glutaraldehyde.
3. Formaldehyde exists in solution as monomers and polymers and it is believed that the polymers are more active at cross-linking. The polymers are present in higher numbers in more concentrated solutions of formaldehyde. The polymers also form when formaldehyde solutions are cooled to 4ÁC. It is possible, therefore, that short fixation times can be used with high concentrations of formaldehyde.

The aldehyde solutions are usually buffered to a neutral pH and we routinely use 100-200 mM concentrations of phosphate or HEPES buffer for this purpose.

Practical Details

Tissue pieces should be small and preferably have been fixed by perfusion. The tissue should be handled carefully so that ultrastructural damage does not occur from squeezing, pulling or shaking. Additionally, the material can be fixed in formaldehyde alone. We routinely use 4% formaldehyde in sodium phosphate buffer (pH 7.4), but other buffers will work. Formaldehyde-fixed material should be stored and transported in formaldehyde.

Cell suspensions can be fixed, in suspension, by the addition of an amount of double strength fixative equal to the volume of culture medium. The presence of extracellular proteins (eg fetal calf serum) does not normally affect intracellular labeling. The cells can be either transported in suspension or pelleted down by centrifugation. If pelleted as soon as the fixative is added and left to fix as a pellet, cell suspensions will normally remain as a pellet.

Cells grown on culture dishes, or in flasks, can also be fixed by the addition of double strength fixative to the culture medium. The cells can then be carefully scraped using a soft wooden stick or piece of Teflon that has been bevelled on one edge to form a blade. The scraped cells can then be transferred to centrifuge tubes and pelleted. Pelleting of small numbers of cells is best performed in the presence of a small amount of protein in the solution (BSA or FCS will work). The protein will stop the cells sticking to the sides of the tube during centrifugation.

An alternative way of removing cells from culture dishes is to treat them with proteinase K. Be aware,however, that this may result in the removal of surface antigens. The cell monolayer is treated, on ice for a few minutes, with 20-50 µg/ml proteinase K in PBS. For most cell types the lower concentration works well. Cool the solution and cells on ice, mix the two and gently pipette the proteinase K solution up and down until cell free areas appear on the plastic. The solution, which will become turbid, can be removed and centrifuged. Some fixative should be added to the cell suspension prior to centrifugation (to preserve the morphology) and a protease inhibitor can be added (PMSF works well) to inhibit the proteinase K activity.

The choice of aldehyde to use as a fixative, and its concentration will depend upon the antigen being studied. However, we routinely use glutaraldehyde or formaldehyde, or mixtures of the two. These are dissolved in sodium phosphate buffer (100-200mM, pH 7.4) or HEPES buffer (same molarity and pH) but other buffers can be used. We routinely use glutaraldehyde at concentrations up to and including 0.5% but higher concentrations can be used. Fixation time will depend upon the tissue and its size but small pieces of perfusion fixed material will be adequately fixed after about 1hr. Glutaraldehyde-fixed material can be transported in buffer without problem. Leaving tissue in glutaraldehyde for longer than 2 hours will make it brittle and difficult to cryosection.

Formaldehyde crosslinking is partly reversible so extensive washing steps after fixation should be avoided. One protocol for formaldehyde fixation that has worked in our hands is to fix in 4% formaldehyde (made from paraformaldehyde powder) for 30-60 min and then overnight in 8% formaldehyde.

Next step: Cryoprotection and Freezing.

Ahmanson Center for
Advanced Electron Microscopy and Imaging