Main Points:
Freshly harvested tissue of interest should be immediately fixed to avoid degradation.
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.
Optimal fixation is key to best histopathology results.
Introduction to Tissue Fixation
The basic testing in anatomic pathology and microscopic examination of tissue requires optimal fixation, processing, sectioning and staining of tissue. Fixation is a critical initial step in histology. Poor fixation can lead to multiple inaccurate results including special stains, immunohistochemistry and other histological techniques. A well-preserved tissue retains its structure and reactivity to reagents like special stains, antibodies for immunohistochemistry and nucleic acid probes for in situ hybridization assays. Both under-fixation as well as over-fixation can cause a great deal of problems in histology, however, under-fixation can be a greater issue than over-fixation and must be avoided. Fixation of fatty tissue like human, rat or mouse brain tissue may need special attention. An optimal tissue fixation ensures preservation of cellular and extracellular structures in order to achieve thin sectioning, optimal histochemical staining and long-term preservation and archival. Here we briefly discuss the important factors for achieving optimal fixation for both human and veterinary tissue.
What is tissue fixation?
Fixation is a chemical process by which biological tissue is preserved to represent the sample’s in vivo state as much as possible. In order to preserve a tissue sample in a state as near to life as possible, the postmortem processes of autolysis, which is self-degradation via proteolytic enzymes, and/or putrefaction, which is the decay of organic matter through microorganism action, must be halted. An ideal fixative should preserve the given tissue sample in a way that is representative of its in vivo situation; cellular and extracellular morphology should be preserved, and the fixative should not denature proteins that are important for histopathological analysis.
Common Fixatives for Histology
A number of fixatives exists and the use of a particular type is dictated by the downstream analysis. For histology, the most effective and commonly used fixatives are aldehyde-based. The following fixatives are recommended for H&E staining, and most IHC markers and special stains:
Neutral Buffered Formalin (NBF): A 10% formaldehyde buffer solution, pH 7.0-7.4 is commonly used in most laboratories. Immediately after surgery, tissue is completely immersed in 10% NBF solution and timed. A ready-to-use solution is available from various vendors in the US. The timing of fixation determines optimal fixation as discussed further.
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.
Size of tissue and volume of fixative
Fixatives are poor buffers; therefore, the pH of solution tends to change during the process of fixation. A large volume of fixative will assure an optimal fixation. The volume of fixative used should be approximately 15-20x the volume of the tissue. A large volume will tend to perform fixation adequately while keeping the reagent stable. For best results, tissue should be cut to into pieces that are no more than 4-5 mm thick in one dimension. If the tissue of interest is needed for other purposes/applications (molecular tests etc.), it can be cut and snap-frozen separately before fixation.
Summary
Fixation must be performed immediately after surgery/dissection.
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.
Written by: Hannah Bashar
Edited by: Rajni Sharma, Ph.D.