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Main Points:
10% Neutral Buffered Formalin (NBF) or 4% Paraformaldehyde solution (PFA) are commonly used
for histology. These are effective fixatives for H&E, and the majority of immunohistochemistry
(IHC) markers and special stains.
2. Paraformaldehyde (PFA) solution: Freshly prepared 4% PFA solution produces similar results and is
cost effective. Due to its fast degradation, this solution is prepared fresh each time before use.
The mechanism of action and amount of formaldehyde in both solutions are the same.
Mechanism of Action
The mechanism of action of fixation is through rapidly terminating all ongoing enzymatic reactions and
metabolic activities by denaturing intrinsic biomolecules. In doing this, proteolytic enzymes that would
otherwise digest the tissue sample via autolysis are denatured, and autolytic processes are stopped.
Fixatives also protect the sample from extrinsic damage as they are toxic to most common
microorganisms (bacteria in particular) that may otherwise colonize a tissue sample. In addition, many
fixatives chemically alter the treated tissue to be less palatable to opportunistic microorganisms, thereby
preventing the process of putrefaction.
Formaldehyde’s mechanism of fixation is through cross-linking, or creating covalent chemical bonds,
between amino acid residues, mostly commonly that of amino acid lysine residues (side chain amino
groups of lysine), which result in methylene bridges. Cross-linking of formaldehyde can also occur between
the aminomethylol groups and phenol, indole, and imidazole side chains. Furthermore, formaldehyde acts
on a variety of amino acids, such as lysine, arginine, tyrosine, asparagine, histidine, glutamine, and serine.
Cross-linking fixatives maintain internal structures of a sample and do not harm the structure of the
protein significantly. The use of formaldehyde is favorable as it maintains morphology of the tissue sample
and secondary and tertiary protein structure are unaffected and thus preserved. It has been proposed
that formaldehyde is an effective fixative because of its fast penetration speed.
Modes of fixation
There are two ways of fixing tissue - immersion and transcardial perfusion. Immersion fixation involves
placing freshly harvested tissue in an adequate amount of fixative. This is the simplest and the most
common way of fixation. Transcardial perfusion, on the other hand, uses the circulatory system to spread
the fixative, which when performed skillfully results in rapid and effective fixation. This technique generally
results in well-preserved morphology with minimum degradation caused due to autolysis or putrefaction.
For rodents and other small animals, transcardial perfusion is highly recommended for obtaining the best
results. Following transcardial perfusion, harvested organ(s) of interest can be immersed in the fixative to
ensure complete fixation.
Length of Fixation
A fixative should be exposed to the tissue sample for as long as is needed for the solution to completely
penetrate the sample. For immersion fixation, certain factors such as density of the tissue sample, rate of
penetration, and temperature must be taken into consideration. It is important to note that rate of
penetration and rate of fixation are two completely different processes of a fixative, with the latter
proceeding slower than the former. A general rule of thumb to apply for the rate of penetration is 1
mm/hour. A fixation time of 24 hours is recommended for NBF-treated samples.
Under-fixation (early withdrawal of the fixative from the treated tissue) can lead to poor morphological
preservation, while over-fixation (late withdrawal of the fixative from the treated tissue) can lead to
fixation artifacts, loss of signal, or increased nonspecific background signals (“noise”). Generally, it is not
recommended to fix the tissue for more than 36 hours to avoid over-fixation. Both are problems that
require their own solutions and must be avoided when fixing a tissue sample. The duration of the
exposure of a sample to the fixative is thus a very important issue that must be carefully calibrated.
After the process of fixation, artifacts may be introduced as the sample dries. These artifacts can be in the
form of organelle-loss, nuclear-shrinkage, and artifactual clumping, so it is vital to keep the treated sample
moist with phosphate buffer/saline solution to continue to accurately preserve the sample.
Summary
Fix tissue in 10% neutral buffer formaldehyde (NBF) solution or freshly prepared 4%
paraformaldehyde solution. Formaldehyde-based fixatives are preferred for long term tissue
preservation and are known to produce best results for sectioning, morphology (H&E), special
stains and immunohistochemistry.
Cut tissue into smaller pieces (max. 4-5 mm), and use ample amount of fixative, making sure tissue
is completely immersed in the fixative.
Fixation must be performed for no more than 24-36 hours depending on the size of tissue. Timing
of the exposure of a sample to the fixative is important and must be calibrated.
Fixed tissue should be transferred to PBS or 70% ethanol and sent for processing to prepare tissue
blocks.
Please consult lab staff if you need to store fixed tissue for a longer time or need additional advise
on fixation and/or shipping tissue samples. Questions can be emailed to info@ndbbio.com.