Fixation (histology)
It terminates any ongoing biochemical reactions and may also increase the treated tissues' mechanical strength or stability.
This allows the investigation of the tissues' structure, which is determined by the shapes and sizes of such macromolecules (in and around cells) as proteins and nucleic acids.
In addition, many fixatives chemically alter the fixed material to make it less palatable (either indigestible or toxic) to opportunistic microorganisms.
Finally, fixatives often alter the cells or tissues on a molecular level to increase their mechanical strength or stability.
This increased strength and rigidity can help preserve the morphology (shape and structure) of the sample as it is processed for further analysis.
Even the most careful fixation does alter the sample and introduce artifacts that can interfere with interpretation of cellular ultrastructure.
A prominent example is the bacterial mesosome, which was thought to be an organelle in gram-positive bacteria in the 1970s, but was later shown by new techniques developed for electron microscopy to be simply an artifact of chemical fixation.
Using the innate circulatory system, the fixative is distributed throughout the entire body, and the tissue doesn't die until it is fixed.
When this method is used, a drainage port must also be added somewhere in the circulatory system to account for the addition of the volume of the fixative and buffer, this is typically done in the right atrium.
Using perfusion has the advantage of preserving morphology,[9] but the disadvantages are that the subject dies and the volume of fixative needed for larger organisms is high, potentially raising costs.
It is possible to decrease the necessary volume of fluid to perform a perfusion fixation by pinching off arteries that feed tissues not of interest to the research involved.
Perfusion fixation is commonly used to image brain, lung, and kidney tissues in rodents, and is also used in performing autopsies in humans.
Preservation of transient or fine cytoskeletal structure such as contractions during embryonic differentiation waves is best achieved by a pretreatment using microwaves before the addition of a cross linking fixative.
Formaldehyde fixes tissue by cross-linking the proteins, primarily the residues of the basic amino acid lysine.
It causes rapid and irreversible changes, is well suited for electron microscopy, works well at 4 °C, and gives the best overall cytoplasmic and nuclear detail.
Protein-denaturing methanol, ethanol and acetone are rarely used alone for fixing blocks unless studying nucleic acids.
The oxidizing fixatives can react with the side chains of proteins and other biomolecules, allowing the formation of crosslinks that stabilize tissue structure.
Mercurials such as B-5 and Zenker's fixative have an unknown mechanism that increases staining brightness and give excellent nuclear detail.
Hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE) gives formalin-like morphology, excellent preservation of protein antigens for immunohistochemistry and enzyme histochemistry, good RNA and DNA yields and absence of crosslinking proteins.
