The first step in the histological process is the isolation and fixation of the desired tissue. Fixation is a chemical process by which biological tissues are preserved from decay, preventing autolysis or putrefaction. The purpose of fixation is to preserve tissues permanently in as life-like a state as possible. Fixation terminates any ongoing biochemical reactions, and may also increase the mechanical strength or stability of the treated tissues. Fixation should be carried out as soon as possible after removal of the tissues to prevent autolysis.
Choosing a Fixative
There is no perfect fixative, though formaldehyde is generally ideal. A variety of fixatives can be used, depending on the type of tissue present and features to be demonstrated. The five major groups of fixatives, classified according to mechanism of action, include: Aldehydes, Mercurials, Alcohols, Oxidizing agents, and Picrates. The most commonly used fixative is 10% buffered-formalin, as it is the most forgiving of all fixatives when conditions are not ideal, and there is no tissue that it will harm significantly. See below for a description of each fixative:
10% buffered-formalin (4% buffered-formaldehyde) is the most widely-employed and universal fixative, particularly for routine paraffin-embedded sections. It is a gas with a strong odour, soluble in water to a maximum extent of 40% by weight and is sold as such under the name of formaldehyde (40%) or formalin (a colorless liquid). 10% buffered neutral formalin preserves a wide range of tissues and is a ‘forgiving fixative,’ in that it requires a relatively short fixation time and can also be used for long-term storage (as it produces no deleterious effects on tissue morphology with nuclear and cytoplasmic detail being adequately preserved). To ensure that tissue is fixed properly, be sure that the 10% buffered-formalin used is not expired. 10% buffered-formalin can be purchased in pre-filled vials for ease of use.
Formaldehyde can also be obtained in a stable solid form, composed of high molecular weight polymers known as paraformaldehyde. Heated paraformaldehyde generates pure gaseous formaldehyde which, when dissolved in water, reverts mostly to the monomer form. 10% buffered-formalin is prepared by dissolving paraformaldehyde in phosphate-buffered saline (PBS) and adjusting the final Ph to 7. It is best to use freshly-made 4% paraformaldehyde each time that tissue is fixed.
Bouin solution, a rapid-penetrating fixative, is comprised of picric acid, acetic acid, and formaldehyde in an aqueous solution. It is not a good fixative when tissue ultrastructure must be preserved for electron microscopy; however, it is a good fixative when tissue structure with a soft and delicate texture must be preserved. Formalin-fixed tissue is normally mordanted with Bouin solution for better staining results for trichrome stains. The acetic acid in this fixative lyses red blood cells and dissolves small iron and calcium deposits in tissue. A variant in which the acetic acid is replaced with formic acid can be used for both fixation of tissue and decalcification. The tissue hardening effect of formalin is balanced by the soft tissue fixation of picric acid. The tissue swelling effect of acetic acid is balanced by the tissue shrinking effect of picric acid. When using Bouin solution, several potential problems can arise. Due to the formalin in the solution, formalin pigment may be present when viewing tissue sections under the microscope. Wet tissue should be fixed in Bouin solution for less than 24 hours, ideally between 4-8 hours. Excess picric acid should be washed out of tissue using several alcohol and water solutions or staining quality may deteriorate over time. Wet tissue fixed in Bouin solution should be stored in an alcohol and water solution rather than Bouin solution. Since Bouin solution contains formaldehyde, picric acid, and acetic acid, appropriate safety precautions should be taken. In particular, noting that picric acid can be explosive, sensitive to friction and shock when dry, and can form unstable metal picrates when in contact with some metals.
Glyoxal is the simplest dialdehyde and, like formaldehyde, it readily forms hydrates and polymers. Decomposition occurs rapidly in neutral or alkaline solutions, so aqueous solutions must be buffered to ph4 to be stable, and they must also contain a small proportion of ethanol, which catalyzes the reaction of Glyoxal with proteins. Addition to and cross-linking of proteins is quicker than the equivalent reactions of formaldehyde. Adequate fixation of small specimens by Glyoxal is achieved in approximately one hour. The Glyoxal-based fixatives available to laboratories are generally sold as pre-made solutions with trade names and undisclosed compositions. Proprietary Glyoxal fixatives are reported to be useful for general histology and immunohistochemistry.
If you have questions regarding tissue fixation, please contact Wax-it Histology Services via email at firstname.lastname@example.org or by phone at 604.822.1595.
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