Overview: The first step in the histological process is the isolation and fixation of the desired tissue. The purpose of fixation is to preserve tissues permanently in as life-like a state as possible. Fixation should be carried out as soon as possible after removal of the tissues to prevent autolysis. There is no perfect fixative, though formaldehyde comes the closest. Thus, a variety of fixatives are available for use, depending on the type of tissue present and features to be demonstrated. The five major groups of fixatives, classified according to mechanism of action, include: Aldehydes, Mercurials, Alcohols, Oxidizing agents, and Picrates. We use 10% buffered-formalin for most of our tissues as it is the most forgiving of all fixatives when conditions are not ideal, and there is no tissue that it will harm significantly.
- 10% Buffered formalin (Fisher Cat No. SF100-20)
There are two ways to fix the organs of interest:
1) The first requires you to harvest the organs fresh and place immediately into fix (Very important to get the tissue into the fixative ASAP) . You can store the fix in an appropriately sized container. With this method it helps to cut the tissue into smaller pieces to ensure rapid penetration of fix to the central regions, but do not do this if you are interested in whole sections or organs. Tissues such as heart, if you can, nick a piece from the bottom allowing fixative to penetrate into the tissue.
2) Alternatively you may want perform a perfusion fixation. This method is more time consuming but allows for a more immediate and deeper penetration of fix. This is usually performed using a syringe and applying a very small amount of pressure via needle entry into either the left ventricle of the heart or the Aorta (puncture the right atrium to allow flow of fix) to target all organs. Often PBS is injected for a short while prior to the fix in order to remove blood. If the infusion is done correctly, the brain will be white in color and free 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 fix.
The volume of fix should be at least 10X the tissue volume (more is better). At this stage you may bring the tissues to us or you may continue to follow the outlined procedure.
Place the tissue in fix and leave at room temperature all day (~6hrs). Change the fix, and transfer to 4oC overnight continuing to change fix daily. Allow for 24 -72hrs for proper fixation; with smaller tissues the time is less (16-24hrs). After 1-3 days, when it is properly fixed, the sample can be placed in 70%EtOH for long-term storage, still maintained at 4oC. Do not store in PBS after fixing as the fixation procedure is reversible in PBS.
For more suggestions or advice on how to best collect and fix your tissue samples, read our sample preparation for frozen applications and fixation and shipping protocol for FFPE services for more in depth details.
You can drop your samples off at room 202 of the Gerald McGavin Building, located near the Life Sciences building. Buzz us on the board under Wax-it Histology Services.
If you are shipping, make sure to fill each container to the top (either with formalin or 70% EtOH) allowing no air so that the tissue is not going to get knocked around during shipping. Also make sure to seal with parafilm around the lid. So far we have not had any problems with samples shipping across the border (US and Canada) with 10% buffered-formalin, 70% EtOH or as frozen blocks on dry ice. If you have specific questions regarding shipping, please do not hesitate to contact us for more information.