All samples used in the IHC / ICC experiment must be Fixing Agent to maintain tissue morphology and retain the antigenicity of the molecule of interest. Fixing Agent changes the chemical composition of the tissue, so there is often a trade-off between maintaining the tissue structure and preserving the epitopes.
Incomplete fixation (indefinite) of cells or tissues may result in rapid hydrolysis of the target protein within the tissue and reduced specific immunoreactivity. However, excessive fixation (Fixing Agent overgrowth) may result in epithelial masking or strong nonspecific background staining, which may mask specific markers.
The ideal fixative is, of course, the best to maintain the morphology of cells and tissues, and to maximize the preservation of the immune activity of the antigen. However, there is no standard immobilization solution to date for a variety of antigen fixation. After fixation with the same fixative, different tissues may exhibit distinct staining results. Therefore, different antigens and samples must be tested repeatedly to select the best fixative.
Formaldehyde is the most common fixative for tissue and intracellular protein targets. Formaldehyde-mediated tissue fixation is thought to depend on the formation of protein-protein and protein-nucleic acid cross-linking with 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 fixative. The excessive fixation of formaldehyde modifies the amino acids (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 the appropriate alternative.
★ Note: Formaldehyde solution should be dispensed and frozen, or stored at 4-8 ° C for no more than a month.
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! Thus, they compete with water for protein hydrogen bonds that 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 fixatives. Alcohols do not penetrate like formaldehyde and are mainly used to immobilize 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.
Acetone is a strong dehydrating agent that can cause irreversible precipitation of tissue proteins. It is often used for slices that are not Fixing Agent, fast frozen tissue.
Organization of the Fixing Agent
In the study of the integrity of small animals such as mice, rats and guinea pigs, perfusion fixation of the entire animal is the best way to preserve the antigen. It involves the use of a fixative to replace the animal's whole blood. However, the whole animal's perfusion is not enough to fix the target tissue. In these cases, the anatomical tissue can be infiltrated in the fixative. For example, 4% formaldehyde is a common solution for tissue perfusion and infiltration fixation.
In order to strengthen the penetration of the immobilization process during the infiltration fixation, it is recommended that the thickness of the tissue be not more than 10 mm. For complete fixation, the volume of the fixative should be 50-100 times the volume of the tissue. The fixation is usually carried out at room temperature for 4-24 hours. It is important to optimize the fixation conditions because of insufficient fixation or excessive fixation that may reduce or disrupt the immunoreactivity of the tissue.
Culture of 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.
The immobilization of cultured cells is usually only the removal of the medium and the addition of the fixative. However, changes in surface tension after removal of the culture medium may impair certain cell types. If this is the case, the fixative 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% fixative. The pre-fixation step makes the cells harder so that they can withstand any possible harmful effects caused by surface tension changes