Immunofluorescence is a very delicate and finicky labeling technique. Every step in the preparation process is crucial. The process begins with the removal of a tissue sample that needs to be studied. Samples must be kept in a state as close to the environment they were removed from as possible, to prevent autolysis and decay to achieve accurate results. This process of preserving the tissue for this purpose is known as ‘fixing’ the tissue. This can be done in a myriad of different ways from heat fixation, to cryofixation, to chemical fixation, and each of these methods use a multitude of different techniques, depending on the final labeling or staining goal. At Visikol, we typically use samples that have been cryofixed or samples fixed in formalin.
Issues Arising from a Delay Between Tissue Removal and Fixation
The moment tissue is removed from the specimen the tissue begins to degrade, and lysosomal enzymes begin to leak into cells. These enzymes destroy cell structures and allow for tissue degradation, meaning the tissue will not adhere to the slide. The study of histology requires that the samples remain in full, and that proteins and antigens remain intact. If improper or poor fixation methods are used, this may not be the case.
If there is a delay between tissue removal and fixation, there are a variety of issues that can arise. Such as-
-The lysosomal enzymes can completely denature any areas of interest on the sample.
-The sample can decay and end up falling apart during processing.
-The paraffin can have trouble penetrating the sample and the tissue can fold and tear during cutting.
A poorly fixed sample showing tissue loss and degradation
Importance of Choosing a Fixation Technique
The fixation technique chosen is also crucial to obtaining accurate results. There are pros and cons to both formalin and cryofixation. The formalin fixation process can be slow, as the formalin needs to completely penetrate the sample and can lead to the issues outlined above. On the other hand, cryofixation can result in ice crystals that cause labeling artifacts and background staining. This step must be taken properly and chosen carefully, as many factors, such as the sample type and the results scientists are looking for will determine the proper method and timing of fixation.
Additional Resources
Visikol and its cutting-edge Multiplex technology utilizes immunofluorescence techniques and standard immunohistochemistry methods which offer a new paradigm in research, opening a whole new door to understanding the tissue microenvironment, disease progression, and treatment. If you’d like to learn more, visit our Multiplex Technology Page, our Overview Page, or our recent blog post about the Clinical Applications of Multiplex Imaging and how it can be utilized as a Clinical Tool.
And of course, please check out Multiplex panel page detailing each of the panels in more detail and please reach out to our team with any questions or to get started on a project!