In some cases, it is not possible to analyze the material immediately after collection. Then a procedure is necessary which permits storage of material for analysis at later date. This is especially critical in experiments involving the measurement of cell cycle kinetics, when samples have to be collected and analyzed at specific time intervals.
Due to changes in chromatin structure, nuclei isolated from fixed tissues are not recommended for determination of DNA content in absolute units (genome size).
1. Fix plant tissues (10 - 100 mg) by addition of 20 ml of formaldehyde fixative for 10 min at 5°C.
The optimal concentration of formaldehyde and the duration of fixation should be determined empirically for given material (to achieve the lowest backround and the highest possible resolution of peaks in the DNA content histograms).
2. Wash out formaldehyde fixative by three changes of Tris buffer, each for 10 min at 5°C.
3. The fixed tissues can be stored at 4°C for up to several days.
4. Homogenize the tissues by crushing with a glass rod in 1ml of ice-cold Tris buffer or LB01 in a petri dish.
Alternatively, nuclei can be isolated by chopping the tissues with a new razor blade and/or scalpel. It is also possible to release the nuclei using a motorized homogenizer (e.g. Polytron PT 1200). In this case, fixed tissues are transferred to a 12 x 75mm polystyrene tube containing ice cold LB01 buffer. This approach is especially convenient for isolation of nuclei from very small root tips and/or small amounts of cultured cells.
5. Filter the suspension through a 42 µm nylon mesh.
6. Store the nuclei at 4°C prior to analysis.
Fixed nuclei can be stored for more than a week. If prolonged storage is required, isolated nuclei should be employed, rather than fixed tissues.
7. Add DAPI to a final concentration of 2 µg/ml.
Binding of propidium iodide to DNA in formaldehyde-fixed chromatin is impaired and the use of DAPI for DNA staining is recommended. Alternatively, the negative effect of the fixation may be reversed by heating (Overton and McCoy 1994) or by acid hydrolysis (unpublished).
8. Analyze relative DNA content of isolated nuclei.
