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Glycerol (a non-fluorescent grade) is somewhat effective as an antifade solution if nothing else is available, although fluorochromes will fade rapidly.
0.5 % p-phenylenediamine (PPD) (Free Base, Sigma) made up in 20 mM Tris, pH 8.8 with 90 % glycerol (non-fluorescent) is a good antifade mountant with most fluorochromes. Store at 4C in the dark. It will go dark brown with time, but this more offends the eye than reduces the effectiveness it seems.
Otherwise, commercial antifade solutions such as Citifluor, VectorShield, or ones from Molecular Probes can be used. Not different fluorochromes respond differently to various antifades (see www.probes.com for some parameters).
- It is very important not to expose seedlings, roots and plants during germination and metaphase-arrest to chemicals and fumes, particularly fixatives (e.g. in a cold room also used for chemical storage) and to use clean labware with tight lids (disposable plastic is ideal), clean forceps, and distilled water.
- Root tips from germinating seeds, and plants grown in controlled conditions, often show waves of cell division that may follow internal or environmental rhythms (e.g. light) or correlate with root length. At certain times, there may be no divisions at all, so it may be helpful to make several fixations.
- Representative times are given. For best results fix material after different times of treatment, experiment with different reagents and check the mitotic index by making chromosome preparations. Treating material for too long in arresting agents, particularly colchicine, results in over-condensation of the metaphase chromosomes which might be desirable for counting chromosomes, but not for in situ hybridization where spatial resolution along chromosomes is wanted.
- Fixative should not be contaminated with water, so careful blotting or an extra rinse in fixative is advised.
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