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How to Fix Plant Tissue?

Published in Plant Biology 3 mins read

To fix plant tissue, transfer it to a vial of 3% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4.

Here's a more detailed explanation:

Fixing plant tissue is a crucial step in various biological and microscopic analyses. It preserves the tissue's structure and prevents degradation, allowing for detailed examination. The following outlines the standard procedure and considerations for fixing plant tissue effectively:

Procedure:

  1. Preparation of Fixative: Prepare a fixative solution consisting of 3% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4. The glutaraldehyde acts as the primary fixative, cross-linking proteins and stabilizing cellular structures. The cacodylate buffer maintains a stable pH, essential for optimal fixation.

  2. Tissue Transfer: Carefully transfer the plant tissue sample into the prepared fixative solution. Ensure the tissue is fully submerged to allow for complete and uniform fixation. The size of the tissue sample should be considered; smaller pieces fix more rapidly and evenly.

  3. Fixation Time: Allow the tissue to fix for a duration of 2 hours to overnight. The precise fixation time depends on the type of tissue, its density, and the specific downstream application. Overnight fixation is often preferred for thicker or denser tissues to ensure adequate penetration of the fixative.

  4. pH Considerations: While a pH of 7.4 is generally suitable, the initial pH of plant vacuoles is typically acidic. Therefore, some researchers opt for a slightly higher pH in the fixative solution, up to 7.8, to better preserve cellular structures in certain plant species or tissues.

  5. Optional Sucrose Addition: Adding sucrose to the buffer is usually unnecessary. However, it can be beneficial when working with senescent (aging) tissue, as it can help to maintain cell turgor and prevent collapse. The optimal concentration of sucrose should be determined empirically.

Additional Notes and Considerations:

  • Buffer Choice: While cacodylate is a common choice, other buffers such as phosphate buffer can also be used. Ensure the chosen buffer is compatible with any subsequent processing steps.
  • Temperature: Fixation is typically carried out at room temperature or at 4°C. Lower temperatures can slow down the fixation process but may improve preservation in some cases.
  • Vacuum Infiltration: For tissues that are difficult to infiltrate with fixative (e.g., tissues with air spaces), vacuum infiltration can be employed to remove air and facilitate penetration.
  • Post-Fixation Washing: After fixation, the tissue should be thoroughly washed in buffer to remove excess glutaraldehyde. This prevents unwanted side reactions in subsequent processing steps.
  • Safety: Glutaraldehyde is a hazardous chemical. Always wear appropriate personal protective equipment (gloves, eye protection, lab coat) when handling it and work in a well-ventilated area.

By following these guidelines, you can effectively fix plant tissue for subsequent analysis, ensuring accurate and reliable results.