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| Fixation is the process by which you kill cells and
preserve their contents. No fixative is perfect, however, a lot is known about the various
fixatives that can be used. This Link(http://www.vpp.vet.uga.edu/emlab/manual.html) may take you to the University of Georgia Microscopy Manual. It has details for preparing many fixatives as well as other procedures. The most gentle plant fixative if Formaldehyde prepared from Paraformaldehyde poweder. Commercial Formalin contains some alcohol & this will cause more extraction than pure formaldehyde. The fixative I use contains 4% Formaldehyde (from Paraformaldehyde) dissolved in dilute NaOH with heat. The vessel containg the mixture must be stirred, and heated to the point of boiling in a Fume Hood or well ventilated place. The vessel must be covered to prevent the loss of formaldehyde vapors! Some time ago a colleague tried to dissolve Paraformaldehyde in a Microwave Oven! They had to evacuate the building! I use HEPES or Phosphate Buffer (0.1 Molar) to maintain the pH around 7.4. I use 10% DMSO (Dimethylsulfoxide) and 1% Tween (20 or 80) if I plan to use the Freezing Microtome. DMSO acts as an antifreeze which prevents large ice crystal formation during freezing. It is used in the Cryopreservation of shoot apical meristems. It also penetrates the intercellular spaces and along with Tween (detergent) helps trapped air to escape. DMSO & Tween will cause some extraction so look at fresh sections if you can. Store fixed samples in a refigerator if you can. Most samples will retain their natural pigmentation for months under these conditions. This will vary, of course. |
