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.