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.
The article ‘Fixing Tissue for Optimal Results: Part 1’ outlined a few common fixatives and their best uses, according to the type of tissue present and features to be demonstrated. This article will explore considerations for fixation related to a number of factors including: staining method, length of fixation, promptness and volume of fixative, fixing an organ of interest, and shipping and packaging.
Formalin-based fixatives are generally the least harsh and lend to better types of stains. For immunohistochemistry (IHC), 10% buffered formalin or 4% paraformaldehyde is ideal to ensure proper fixation which allows antigen-retrieval. When using Bouin’s, the effects of the three chemicals present in the solution balance each other; Formalin causes cytoplasm to become basophilic but this effect is balanced by picric acid. This results in excellent nuclear and cytoplasmic H&E staining; however, Bouin’s is a harsher fixative and can affect the staining protocol, and can also lead to potential issues with background staining.
Length of Fixation
Proper tissue fixation depends on the thickness of tissue, since levels of penetration vary from fixative to fixative. Fixatives penetrate tissues at different rates and these rates can be further affected by heat. Fixation with 10% buffered formalin at room temperature usually provides excellent morphological detail. Adequate fixation time is critical for accurate morphology. It is important not to ‘over-fix’ tissue, as it could become brittle and hard to work with, which could complicate sectioning and impair staining. Alternatively, ‘under-fixing’ will result in tissue that is too soft. Under-fixation can also be associated with artifacts secondary to alcohol exposure during tissue processing, or tissue damage during decalcification procedures. For most tissues, a minimum of 24 hours at room temperature is recommended; however, certain tissues (such as some bloody or fatty tissues) could require longer fixation. It is generally best to fix in formalin or PFA for 24-48 hours and in Bouins for no more than 2-4 hours. In the case of general histology (H&E and or special stains), but not immunohistochemistry, tissue can be stored in 10% buffered formalin for an extended period of time.
Promptness & Volume of Fixative
The tissue should be placed in fixative immediately after surgery or necropsy. The more time that elapses between interruption of the blood supply and fixation, the more post-mortem changes will be observed under the microscope. When fixing tissue, be sure to place the tissue in a pre-filled container/vial of fixative rather than adding fixative subsequently; this ensures that the bottom of the tissue is fully immersed in fixative. The tissue is fixed starting at the periphery of the tissue and working inward towards the centre. Formalin will take time to fully penetrate tissue samples, while smaller samples are more easily saturated; prompt fixation is imperative in both scenarios. The volume of fixative should be at least 10x the tissue volume (more is better) with a 20:1 millilitre ratio of fixative to tissue for optimal fixation. Too little fixative will result in tissue damage and too much will create unnecessary waste.
Fixing an Organ of Interest
To fix an organ of interest, first place the tissue into fixative and let it stand at room temperature for approximately 6 hours. Having a labelled jar with adequate fixative ready can minimize time before fixation and prevent mislabelling of the sample. Once this step is complete, change to fresh fixative and store tissue overnight at 4oC; allow for 24-48 hours for proper fixation. Fixative should be changed daily if requiring more than 24 hours; with smaller tissues, allow for 16-24 hours. After 1-2 days, the sample can be placed in 70% EtOH for long-term storage, still maintained at 4oC. Do not store the tissue in PBS after fixing with formalin, as the fixation procedure is reversible in PBS. There are generally two methods that are used when fixing an organ of interest:
1. The first method requires that the fresh organs are immediately harvested, placed into fixative and stored in an appropriately-sized container. When following this method, cutting the tissue into smaller pieces will help to ensure rapid penetration of fixative to the central regions. If you wish to look at whole sections or organ tissues, such as the heart, ‘nick’ a piece from the bottom to allow the fixative to penetrate into the tissue.
2. Alternatively, one can perform a perfusion fixation; this method is more time-consuming but allows for a more immediate and deeper penetration of fixative. Perfusion fixation is performed using a syringe and by applying a very small amount of pressure via needle entry into either the left ventricle of the heart or the Aorta. Be sure to puncture the right atrium to allow for proper flow of fixative to target all organs. Often PBS is injected for a short period prior to the fixative in order to remove blood. If the infusion is conducted correctly, the brain will be white in color and devoid of blood. Perfusion of the lungs is performed via entry into the trachea to maintain the proper structure. After 15-20 minutes, the tissues are isolated as above and placed in fixative.
Shipping & Packaging
When shipping samples, be sure to fill each container to the top (in either fixative or 70% EtOH), allowing no air bubbles (which will prevent the samples from moving during shipping). Formalin-fixed samples should be shipped in a well-sealed container or jar and any areas in which formalin could leak through, such as the lid of the tube or jar, should be sealed with parafilm.
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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About Wax-it Histology Services, Inc.
Wax-it Histology Services, Inc. is a GLP-recognized CRO that offers general histology services (paraffin and frozen), immunohistochemistry services (general IHC, antibody testing/validation, cross-reactivity studies), GLP and non-GLP histopathology for preclinical toxicology studies, and resin/plastic sectioning for implantable medical devices and bone.