Download Crosslinking fixatives

Fixation
Faculty of Applied Medical Sciences
Presented by:
Histopathology Teaching Assistant Walaa Mal
Content
Introduction
Definition
Purpose of fixation
Features of fixation
Characteristics of fixatives
Fixation process
Types of fixation
Types of fixatives
1.
2.
3.
4.
5.
6.
7.
8.
1.
Cross linking fixatives
1.
2.
2.
3.
9.
Aldehydes
Oxidizing agents
Precipitating fixatives
Other fixatives
References
Introduction


Tissue block is taken by biopsy, surgical excision or postmortem.
Numerous techniques can be used to prepare tissue for microscopical
examination depend on:





Structures examined
Nature of examined tissues
Urgency of investigation
Fresh or preserved specimens
Fresh tissue can be examined as:



Smear: e.g. screening of cancer cervix
Frozen section using cryostat as urgent conditions e.g. during surgery
Method: The fresh tissue is rapidly frozen using liquid nitrogen or carbon dioxide.
 Sectioning is carried out in the refrigerated cabinet of a Cryostat which is
an apparatus designed to keep the microtome knife and tissue at a subzero temperature throughout the sectioning.
Definition of Fixation
 In the fields of histology, pathology, and cell biology, fixation is a chemical
process by which biological tissues are preserved from decay, either through
autolysis or putrefaction.
 Fixation terminates any ongoing biochemical reactions, and may also
increases the mechanical strength or stability of the treated tissues.
Purpose of fixation


The purpose of fixation is to preserve a sample of biological material (tissue
or cells) as close to its natural state as possible in the process of preparing
tissue for examination. To achieve this several conditions must usually be
met.
The aims of fixation are:
1.
2.
3.
4.
5.
Prevent postmortem (PM) degeneration
Prevent autolysis. It is effective against hydrolytic enzymes
Stop the bacterial effect
Harden the tissues, as fixation causes coagulation of proteins
Fixation has a mordanting effect, facilitating subsequent staining of
tissues.
Features of fixatives

First, a fixative usually acts to disable intrinsic biomolecules – particularly proteolytic
enzymes – which would otherwise digest or damage the sample.

Second, a fixative will typically protect a sample from extrinsic damage. Fixatives are
toxic to most common microorganisms (bacteria in particular) which might exist in a
tissue sample or which might otherwise colonise the fixed tissue.

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 such as Nuclear
Morphometry System.

Note: 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.
Characteristics of a good fixative
1.
2.
3.
4.
5.
6.
It must kill the cell quickly without shrinkage or swelling
It must penetrate the tissue rapidly
It must inhibit bacterial decay and autolysis
Harden the tissue and render it insensitive to subsequent treatment
as staining
It should allow tissue to be stored for long time
It should be simple to prepare and economical is use.
Fixation process
 Fixation is usually the first stage in a multistep process to prepare a sample of
biological material for microscopy or other analysis.
 Therefore, the choice of fixative and fixation protocol may depend on the
additional processing steps and final analyses that are planned.
 For example, immunohistochemistry utilises antibodies which bind to a
specific protein target. Prolonged fixation can chemically mask these targets
and prevent antibody binding. In these cases, a 'quick fix' method using cold
formalin for around 24 hours is typically used.
Types of fixation

There are generally three types of fixation process:

Heat fixation:
After a smear has been allowed to dry at room temperature, the slide is gripped by tongs or a clothespin
and passed through the flame of a Bunsen burner several times to heat-kill and adhere the organism to the
slide.
Heat-fixation method can be successfully used for preparing Gram-negative and Grampositive bacteria samples for studies

Perfusion:
Fixation via bloodflow. The fixative is injected into the heart with the injection volume matching cardiac
output. The fixative spreads through the entire body, and the tissue doesn't die until it is fixed. This has the
advantage of preserving perfect morphology, but the disadvantages that the subject dies and the cost is high
(because of the volume of fixative needed for larger organisms)

Immersion:
The sample of tissue is immersed in fixative of volume at a minimum of 2/3rds greater than the volume of
the tissue to be fixed. The fixative must diffuse through the tissue in order to fix, so tissue size and density,
as well as the type of fixative must be taken into account. Using a larger sample means it will take longer
for the fixative to reach the deeper tissue.
Types of fixatives
 Crosslinking fixatives
 Aldehydes
 Oxidising agents
 Precipitating fixatives
 Ethanol
 Methanol
 Acetone
 Other fixatives
 Picric acid

Mercuric chloride
Crosslinking fixatives
“Aldehyde”




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.
By far the most commonly used fixative in histology is the crosslinking fixative
formaldehyde (also named formalin).
Formaldehyde is thought to interact primarily with the residues of the basic amino acid
lysine.
Another popular aldehyde for fixation is glutaraldehyde.



It is believed to operate by a similar mechanism to formaldehyde.
As a somewhat larger molecule, glutaraldehyde may not penetrate thicker tissue
specimens as effectively as formaldehyde.
On the other hand, glutaraldehyde may offer a more rigid or tightly linked fixed

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 protect significant amounts of tertiary
structure as well.
Crosslinking fixatives
“Oxidising agents”

The oxidising fixatives can react with various side chains of proteins and other
biomolecules, allowing the formation of crosslinks which stabilise tissue structure.

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.
Precipitating fixatives
“Denaturing fixatives”

Precipitating (or denaturing) fixatives act by reducing the solubility of protein molecules and
(often) by disrupting the hydrophobic interactions which give many proteins their tertiary
structure.

The precipitation and aggregation of proteins is a very different process from the
crosslinking which occurs with the aldehyde fixatives.

The most common precipitating fixatives are ethanol and methanol. Acetone is also used.

Acetic acid is a denaturant that is sometimes used in combination with the other precipitating
fixatives.

The alcohols, by themselves, are known to cause shrinkage of tissue during fixation while
acetic acid alone is associated with tissue swelling; combining the two may result in better
preservation of tissue morphology.
Other fixatives
 Other fixative agents include


Picric acid
Mercuric chloride.
References
1.
2.
Ryter A (1988). "Contribution of new cryomethods to a better knowledge of bacterial anatomy". Ann. Inst.
Pasteur Microbiol. 139 (1): 33–44. PMID 3289587.
Friedrich, CL; D Moyles, TJ Beveridge, REW Hancock (2000). "Antibacterial Action of Structurally
Diverse Cationic Peptides on Gram-Positive Bacteria". Antiomicrobial Agents and Chemotherapy 44 (8):
2086–2092.
Bunsen burner
Covalent chemical bonds
Transmembrane receptor