Molecular Techniques and Methods

Glutaraldehyde-Paraformaldehyde Fixation of Tissue

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION




MATERIALS AND SOLUTIONS

Glutaraldehyde-Paraformaldehyde Fixation Solution (10 ml)
0.25% Glutaraldehyde ---------------------- 0.1 ml of 25% Glutaraldehyde
4% Paraformaldehyde ---------------------- 1.1 ml of 37% Paraformaldehyde
0.1 M Na-Phosphate Buffer (pH 7.2) ------ 1 ml of 1 M Na-Phosphate Buffer
DEPC-treated H2O ------------------------ 7.8 ml




PROCEDURES

1. For fixation, the material should be of a manageable size.
  • Smaller pieces of tissue, allow greater penetration of the fixative, dehydrating solutions, and embedding material.

    2. After subdividing, place the material as well as a label stating the identity for the tissue in small vials with 10-20 times the volume of Glutaraldehyde-Paraformaldehye Fixation Solution per volume of tissue.

    3. Remove air bubbles, which interfere with proper fixation, by placing the unstoppered vials in an aspirator or vacuum dessicator under vacuum.
  • Aspirate for short intervals of time until material sinks to the bottom or just under the surface.
  • Meristematic tissue should be aspirated for a very short interval.
  • Tap the specimen bottle gently to aid in the removal of air bubbles.
  • Very buoyant material should be placed in a tall thin vial and held under the surface with a cheesecloth plug.
  • Most pieces are submerged after vacuuming; usually any floating piece will sink if pushed under the surface.
  • Be sure to aspirate the same type of tissue together; i.e., do not mix highly vacuolate material with meristematic tissue.
  • If a specimen is too large or especially recalcitrant, leave the tissue overnight under vacuum, making certain that enough liquid is present in the vial to cover the tissue.

    4. Fix the tissue at 4oC for 2-3 hours depending on the size of the tissue

    5. Rinse fixed tissue in 0.1 M Na-Phosphate Buffer (pH 7.2) for 30 min. Repeat.

    6. Dehydrate the tissue as follow.

    Step
    Ethanol
    Time
    1
    5%
    30 min
    2
    25%
    30 min
    3
    50%
    30 min




    NOTES




    KIT INFORMATION




    REFERENCES



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