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This page is part of our IHC application guide: download it or read it online.
Fixation of the tissue sample is essential to maintain cell and tissue morphology during the IHC experiment and during storage. It also prevents the autolysis and necrosis of excised tissues, preserves antigenicity and enhances the refractive index of tissue constituents. The fixative used is influenced by the target antigen as well as the desired detection technique (fluorescent or chromogenic). The tissue sample is then either embedded in paraffin or frozen. Embedding is important in preserving tissue morphology and giving the tissue support during sectioning. Some epitopes may not survive harsh fixation or embedding. The tissue is typically cut into thin sections (5-10 µm) or smaller pieces (for whole mount studies) to facilitate further study.
When generating paraffin-embedded tissue samples, the tissue must be fixed before embedding in paraffin. Fixation is achieved by perfusion or immersion immediately following dissection. The process typically takes 4 - 24 hours (fixation for longer than 24 hours is not recommended as it may lead to overfixation, which may mask the antigen).
Standardized fixatives for each type of antigen are essential for reproducible staining - an antigen that has been inappropriately fixed may not be detected. The most suitable fixative for an IHC experiment depends on the antigen, as illustrated in the figure below. Some guidelines for the type of fixative to use are given in the table below.
Effect of fixative on immunostaining patterns.
Crotonylation of Histone H2B K5 is more readily detected in immunocytochemistry of HeLa cells fixed with methanol (right) than cells fixed in formaldehyde (left). Primary: ab177139, rabbit polyclonal to Histone H2B (crotonyl K5), 1 μg/ml. Secondary: ab150081, goat anti-rabbit IgG H&L (Alexa Fluor® 488), 0.5 μg/ml. Nuclei are stained with DAPI and tubulin (red) is stained with ab7291 and ab150120.
|Most proteins, peptides and enzymes of low molecular weight|
Cells / cytological preparations: 4% formaldehyde
Tissue sections: 10% Neutral-Buffered Formalin (NBF)
|Delicate tissue||Bouin's fixative|
|Small molecules such as amino acids||4% formaldehyde|
|Blood-forming organs (liver, spleen, bone marrow)||Zenker’s solution|
|Connective tissue||Helly's solution|
|Nucleic acids||Carnoy’s solution|
|Large protein antigens (e.g., immunoglobulin)||Ice-cold acetone or methanol (100%)|
|Nuclear morphology||Zinc formalin|
|For electron microscopy||4% formaldehyde - 1% glutaraldehyde|
Note that formaldehyde is a gas that retains its chemical properties in aqueous solution. Formalin is a saturated solution of 37-40% w/v formaldehyde in water. 10% formalin is therefore roughly equivalent to 4% formaldehyde. Paraformaldehye is a solid comprised of large polymers of formaldehyde. Formalin contains ~10% methanol, which is added by the manufacturer to slow the polymerization of formaldehyde in solution to paraformaldehyde. As the added methanol can have a negative impact on the fixation of some samples, some protocols recommend making formalin from paraformaldehyde immediately before sample fixation.
After fixation, the tissue is dehydrated to enable embedding with paraffin, which is water-insoluble. The tissue is dehydrated gently by immersion in increasing concentrations of a dehydrating agent such as alcohol. This gradual change in hydrophobicity minimizes cell damage. The dehydrating agent is then cleared by incubation in xylene prior to paraffin embedding. Paraffin is typically heated to 60°C and then allowed to harden overnight. Finally, the tissue is sectioned using a microtome. Tissue sections may be dried by onto microscope slides and stored for extended periods at room temperature. Tissue sections are then rehydrated prior to commencing the immunostaining protocol.
Frozen tissues are prepared by immersing the tissue in liquid nitrogen, isopentane or by burying the sample in dry ice. Snap-freezing is frequently used when detecting post-translation modifications such as phosphorylation. The frozen tissue is cut using a cryostat. The resulting sections can be stored at -80°C for up to 1 year. The frozen tissue sections may then be fixed, typically with an alcohol such as methanol or ethanol. As alcohols do not mask epitopes, their use avoids the need for antigen retrieval.