Скачать или смотреть Types Of Fixative || Tissue Processing || Histology

Types Of Fixative || Tissue Processing || Histology || Part 02 || WBSSC PG Biology|| WBSLST PG Zoolgy
fixation is the preservation of biological tissues from decay due to autolysis or putrefaction. It terminates any ongoing biochemical reactions and may also increase the treated tissues' mechanical strength or stability. Tissue fixation is a critical step in the preparation of histological sections, its broad objective being to preserve cells and tissue components and to do this in such a way as to allow for the preparation of thin, stained sections. 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.
There are generally three types of fixation processes depending on the sample that needs to be fixed.

Heat fixation
Heat fixation is used for the fixation of single cell organisms, most commonly bacteria and archaea. The organisms are typically mixed with water or physiological saline which helps to evenly spread out the sample. Once diluted, the sample is spread onto a microscope slide.

Immersion
Immersion can be used to fix histological samples from a single cell to an entire organism. The sample of tissue is immersed in fixative solution for a set period of time. The fixative solution must have a volume at least 10 times greater than the volume of the tissue.


Perfusion
Perfusion is the passage of fluid through the blood vessels or natural channels of an organ or organism. In tissue fixation via perfusion, the fixative is pumped into the circulatory system, usually through a needle inserted into the left ventricle. This can be done via ultrasound guidance, or by opening the chest cavity of the subject.

Chemical fixation
In both immersion and perfusion fixation processes, chemical fixatives are used to preserve structures in a state (both chemically and structurally) as close to living tissue as possible. This requires a chemical fixative.

Crosslinking fixatives – aldehydes
Crosslinking fixatives act by creating covalent chemical bonds between proteins in tissue. This anchors soluble proteins to the cytoskeleton, and lends additional rigidity to the tissue. 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.

The most commonly used fixative in histology is formaldehyde. It is usually used as a 10% neutral buffered formalin (NBF), that is approx. 3.7%–4.0% formaldehyde in phosphate buffer, pH 7.

Another popular aldehyde for fixation is glutaraldehyde. It operates similarly to formaldehyde, causing the deformation of proteins' α-helices. However glutaraldehyde is a larger molecule than formaldehyde, and so permeates membranes more slowly.

Some fixation protocols call for a combination of formaldehyde and glutaraldehyde so that their respective strengths complement one another.

These crosslinking fixatives, especially formaldehyde, tend to preserve the secondary structure of proteins and may also preserve most tertiary structure.

Precipitating fixatives – alcohols
Precipitating (or denaturing) fixatives act by reducing the solubility of protein molecules and often by disrupting the hydrophobic interactions that give many proteins their tertiary structure. The precipitation and aggregation of proteins is a very different process from the crosslinking that occurs with aldehyde fixatives.

The most common precipitating fixatives are ethanol and methanol. They are commonly used to fix frozen sections and smears. Acetone is also used and has been shown to produce better histological preservation than frozen sections when employed in the Acetone Methylbenzoate Xylene (AMEX) technique.

Protein-denaturing methanol, ethanol and acetone are rarely used alone for fixing blocks unless studying nucleic acids.

Acetic acid is a denaturant that is sometimes used in combination with the other precipitating fixatives, such as Davidson's AFA.

Oxidizing agents
The oxidizing fixatives can react with the side chains of proteins and other biomolecules, allowing the formation of crosslinks that stabilize tissue structure. However they cause extensive denaturation despite preserving fine cell structure and are used mainly as secondary fixatives.

Osmium tetroxide is often used as a secondary fixative when samples are prepared for electron microscopy. (It is not used for light microscopy as it penetrates thick sections of tissue very poorly.)

Potassium dichromate, chromic acid, and potassium permanganate all find use in certain specific histological preparations.

Mercurials
Mercurials such as B-5 and Zenker's fixative have an unknown mechanism that increases staining brightness and give excellent nuclear detail