Automated Microscopy in Forward Genetic Screening of Arabidopsis - Plant Cell Morphogenesis: Methods and Protocols - page 61

3. Caution, DMF is toxic. Avoid direct contact with skin and
perspiration.
4. Triton X-100 is very viscous and foams.
5. Fe salts are hazardous to health. Avoid direct contact with skin
and perspiration. In reacting with acids, K 3 [Fe(CN) 6 ] pro-
duces highly toxic gas.
6. The Fe-salt concentration needed depends upon the strength
of the promoter used and must be optimized for each reporter
line. The Fe salts catalyze the formation of colored precipitate
from the colorless intermediate. This step is not catalyzed by
the GUS, and the presence of Fe salts ensures that the color-
less intermediate does not diffuse from the place of its forma-
tion. In most cases, the concentration of Fe salts varies in the
range 0.5-5 mM.
7. Our protocol is optimized for using a 24-well cell culture plate
(diameter of each well corresponds to 15 mm). Thus, all vol-
umes mentioned in the further text could be subject to opti-
mization if a staining plate of different dimensions were used.
8. Be careful during preparation of clearing solutions. Protect
yourself (wear gloves and goggles) and work in a fume hood.
Caution, both reactions are exothermic! Keep the 7 %
NaOH/60 % ethanol solution at 4 °C in an opaque container
for no more than 1 month or until the yellowish color of the
solution is detectable.
9. Use a cover slip of maximum length 50 mm. The loader in the
automated microscope uses the slide label area to handle the
specimen, and longer cover slips would interfere with that.
This could cause damage to your preparation and/or the
automated microscopy system.
10. Alternatively, a motorized lab tube rotator could be used.
11. The purpose of this cold treatment is to break seed dormancy
(vernalization). Even in the case of using nondormant eco-
types (e.g., ecotype Col-0), however, the cultivation of seeds
at 4 °C synchronizes germination and improves its frequency.
12. Use plastic stands to avoid leakage of condensate from the
Petri dishes during cultivation. The condensate contains
sucrose that would result in heavy contamination of the
growth chamber by fungi.
13. Do not use a strong vacuum source (e.g., an oil pump), as
strong vacuum could damage the specimen.
14. Incubation time is dependent on the strength of the promoter
used in the respective reporter line. It can differ among indi-
vidual reporter lines, growth conditions, tissue types, etc., and
must therefore be optimized. Plants are not fi xed during stain-
ing and in cases of highly sensitive promoters and/or genes,