Fixation is needed in Immunohistochemistry to preserve the tissue samples in a manner that prevents degradation and preserves antigenicity and tissue architecture. Tissue samples can undergo autolysis (self-degradation) or become contaminated with microorganisms, which can result in the loss of antigenicity and tissue integrity. Fixation chemically cross-links proteins in the tissue, preserving the tissue structure and stabilizing the antigens, making it possible to detect them using Immunohistochemical techniques. Fixation helps to preserve the tissue in a consistent state, allowing for reproducible results and comparison of samples over time.
There are two types of fixation: formalin (4% paraformaldehyde) and alcohol-based (such as ethanol or methanol).
Advantages of formalin fixation include: widely used, easily accessible, well-established protocols, and good preservation of antigenicity and tissue architecture.
Disadvantages include: possible modification of antigenic sites and over-fixation can interfere with antigen retrieval methods.
The length of time for formalin fixation of tissue samples in Immunohistochemistry is typically between 4 to 48 hours, but can vary depending on the type of tissue and the intended downstream applications. A general guideline is to fix the tissue samples in 4% paraformaldehyde solution for at least 4 hours, but longer fixation times (up to 24-48 hours) can be used to ensure complete fixation.
It is important to note that over-fixation can interfere with antigen retrieval methods and alter antigenic sites, while under-fixation can result in loss of antigenicity. The optimal fixation time should be determined by trial and error and validated by performing control staining and evaluating the results. Factors such as tissue type, antigenicity, and intended downstream applications should also be taken into consideration when determining the optimal fixation time.
Advantages of alcohol-based fixation include: minimal alteration of antigenic sites, better preservation of some epitopes and faster fixation time.
Disadvantages include: decreased preservation of tissue architecture, potential loss of some antigens, and need for specialized fixation protocols.
The length of time for alcohol-based fixation of tissue samples in Immunohistochemistry can vary depending on the type of tissue and the intended downstream applications, but is typically much shorter than formalin fixation. Alcohol-based fixation, such as ethanol or methanol, is often performed for 5-10 minutes at room temperature or for 30 minutes at -20°C.
It is important to note that the optimal fixation time for alcohol-based fixation should be determined by trial and error and validated by performing control staining and evaluating the results. Factors such as tissue type, antigenicity, and intended downstream applications should also be taken into consideration when determining the optimal fixation time.
It is important to use a sufficient amount of alcohol-based fixative to preserve the tissue, and to avoid over-fixation, which can interfere with antigen retrieval methods and alter antigenic sites.
Things to Note:
The choice of fixation method depends on the type of tissue and the antigen being studied, as well as the intended downstream applications.