It is best to maintain the cell and tissue morphology, and to maximize the preservation of the immune activity of the antigen. However, none of the standard Fixing Agents has so far been used for a variety of antigen fixation. After fixation with the same Fixing Agent, different tissues may exhibit distinct staining results. Therefore, different antigens and samples must be tested repeatedly to select the best Fixing Agent.
Formaldehyde is the most common Fixing Agent for tissue and intracellular protein targets. Formaldehyde-mediated tissue fixation is thought to be dependent on the protein-protein and the formation of a protein-nucleic acid cross-linked with a methylene bridge (-CH2-). Formaldehyde can chemically link NH2 (amino) and CONH (peptide) groups, NH2 and NH, or NH2 and NH2 groups.
Formaldehyde is a good choice for most IHC / ICC applications, but is not a universal Fixing Agent. The excessive fixation of formaldehyde modifies the amino acid (part of the epitope) and blocks the binding of the antibody. However, in most cases, epitopes can be exposed using antigen repair techniques to restore antibody binding. The study also showed that formaldehyde induces phosphorylation-dependent epitope translocation from the cell membrane to the cytoplasm. In this case, ice pre-cooled anhydrous methanol or anhydrous ethanol is an appropriate alternative.
The most commonly used cells and tissue immobilized alcohols are methanol and ethanol. The molecular structure of methanol and ethanol is similar to that of water! Therefore, they compete with water for protein hydrogen bonds to replace water molecules in tissues. This reduces the protein's constant at the isoelectric point by reducing the dielectric constant of the protein and blocks the binding of the antibody-epitope due to conformational changes. Although alcohols affect the tertiary structure of proteins by interrupting hydrogen bonds, they seem to stabilize the secondary structure of the protein.
However, it is generally believed that alcohols do not retain tissue morphology like formaldehyde Fixing Agents. Alcohols do not penetrate like formaldehyde, mainly used to fix frozen tissue sections and cells. Therefore, alcohol fixation is more suitable for membrane surface antigens. Antigen repair is not recommended after alcohol fixation, as it is generally considered too severe and may impair the integrity of tissue sections or cells.
Unlike tissue samples, the fixation time of the cultured cells is shorter and the concentration of the fixation solution is lower. For example, with 2% formaldehyde solution at room temperature for 20 minutes, often enough to retain the cell morphology and antigenicity.
Culture of cells is usually fixed only to remove the medium, and can be added to the fixed solution. However, changes in surface tension after removal of the culture medium may impair certain cell types. If this is the case, the Fixing Agent may be added directly to the medium. For example, by adding 4% formaldehyde to the same volume as the culture medium, a 2% formaldehyde solution will be obtained which is sufficient to pre-fix the cells. After 2 minutes, the pre-fixation medium should be replaced with a fresh 2% Fixing Agent. The pre-fixation step makes the cells harder so that they can withstand any possible harmful effects caused by surface tension changes