The Importance of Dehydration and Rehydration of Fixed Tissue for Immunolabeling

In order to optimize the quality of microscopic images obtained from Formalin Fixed Paraffin Embedded (FFPE) samples, the starting condition of the fixed tissue must mimic the organic tissue as closely as possible. In order to achieve this, the tissue must first be fixed as soon as possible after removal from the specimen. Fixing the tissue properly preserves it from decay and halts all ongoing biochemical reactions.

Rehydrated Kidney Sample

 A properly de- and rehydrated kidney sample labeled for α-Tubulin (green) and nuclei (blue)

After fixation, the tissue must be dehydrated to allow for complete penetration of the tissue by the paraffin wax. Since wax is not soluble in water, but is soluble in xylene, the water in the tissue must be replaced with xylene. Unfortunately, xylene is also not soluble in water but is soluble in alcohol. The sample must be gently dehydrated by immersing the samples in a graded series of alcohol solutions, ending with pure 100% reagent or isopropyl alcohol and then xylene. If one were to simply immerse the sample in 100% alcohol, degradation of the sample and proteins could occur. Improper dehydration can result in incomplete saturation of the sample with paraffin wax and, consequently, softness of the tissue.

Softness of the tissue can lead to uneven slices, tears, or ripples in the tissue and suboptimal, or even unusable, images after labeling or staining. After a usable slice of tissue is obtained, ironically, the tissue must be rehydrated. Most staining and labeling solutions and protocols are aqueous and thus, the wax must be removed again from the sample mounted on the slide. This process is essentially the opposite of the dehydration process and, when done properly, allows for complete infiltration of the sample with the chosen stain or immunolabel. If this process is done improperly, this can result in incomplete infiltration and as a result a false negative result.

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.

Please reach out to a remember of our team if you’re interested in learning more about Multiplexing technology.

2022-08-10T08:06:56-05:00Tags: , |

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