Fixation is a treatment that scientists use to preserve cells or pieces of tissue so they can be analyzed under a microscope. Fixation allows the cells or tissue to be stored for months while unfixed cells can only remain stable for hours or days. Fixation also "freezes" the cell so all proteins and other cellular components are immobile and the cellular structure does not change. Once the cells are fixed they are often analyzed by staining them or by a technique called immunofluorescence. In immunofluorescence an antibody with a fluorescent tag is used to bind to a certain protein in the cell. The cellular location of the protein can then be determined by looking for fluorescent areas under a microscope. Fixation allows the antibody to move freely through the cell to locate the protein.
Three different fixatives can be used to fix cells:
- Cross-Linking Reagents: These reagents freeze components like proteins and nucleic acids in place by linking them together. This is done by forming covalent bonds between their free amine groups. Formaldehyde is the most common reagent, but glutaraldehyde can also be used. These reagents do not affect lipids and leave the cell membrane intact. Therefore, an additional step is required where a detergent such as Triton-X is added to permeabilize the cell membrane so the antibody can get into the cell. The main downside to this fixative is that it may directly alter the protein of interest and prevent the antibody from binding to it.
- Organic Solvents: Solvents such as acetone, acids, and alcohols such as methanol are able to dehydrate the cell and remove its lipids, including those found in the cell membrane. They also precipitate the proteins, making them easier to be accessed by the antibodies. These fixatives work quickly and do not require the additional permeabilization step like the cross-linking reagents do. They also do not affect the antibody-protein interaction because they do not alter proteins.
- Cryofixation: Here the cells or tissue are rapidly frozen by placing them in liquid nitrogen or freon. This technique is the best method for preserving cells, however it is uncommon since most laboratories do not keep the required equipment.
It is important to choose the right type of fixative. For example, certain fixatives such as methanol cannot penetrate very far and therefore can only be used on thin samples. The best fixative also depends on the type of cell and where the protein of interest is located in the cell. Organic solvents work best on proteins that are located on the cell surface, while cross-linking agents work better on proteins that are inside the cell. Researchers often test several different types of fixatives to determine which gives the best immunofluorescence signal.
- Current Protocols in Cell Biology, Volume 1, 2001