Does anyone have experience with high pressure fixation regimes for plant tissues with different vacuolation degrees. Primarily I would like to apply HPF to Arabidopsis roots, but I would greatly appreciate any input regarding hypocotyls whereby cells are more vacuolated and additionally are surrounded by waxy cuticle that may hamper temperature reduction rate necessary for vitreous ice formation. Further, I would like to ask for protocols of freeze substitution for either structural preservation and/or immunodetection at TEM level
Dear Komis
The roots of Arabidopsis thaliana are very difficult to manipulate to achieve good histological preparations, the thick cell wall and intracellular vacuolation make it even more difficult to cut microtome.
I solved by developing a fixative that is normally used in botany the FAA and I have added the 2% glutaraldehyde. The glutaraldehyde completely infiltrates the radical tissue also penetrating inside the vacuoles in this way the roots tissue assumes a degree of compactness homogeneous and can be effectively processed for histological analysis and also maintaining a structural morphology without artefacts.
Best regards
Fabio
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