Automated Microscopy in Forward Genetic Screening of Arabidopsis - Plant Cell Morphogenesis: Methods and Protocols - page 57

2. Use soft tweezers to transfer seedlings from a Petri dish with
MS media into the staining plate so that each well contains
approximately 30 seedlings from the particular M2 family.
3. Place the cultivation plate under gentle vacuum in the desicca-
tor for 10 min ( see Note 13 ).
4. Incubate the seedlings in the GUS staining buffer for 8 h at
37 °C in darkness ( see Note 14 ).

3.3 Sample Clearing

This process allows obtaining a transparent specimen that is suit-
able for differential interference contrast (DIC) microscopy and is
compatible with GUS-stained tissue [ 9 ]. In addition to seedlings,
it can be used for other tissues from the later stages of development
(e.g., fl owers or infl orescence stems). In the fi rst step, the chloro-
phyll is removed and the material is fi xed. In the following steps,
the sample is rehydrated in a decreasing ethanol series and satu-
rated by 50 % glycerol. Unless otherwise specifi ed, use laboratory
temperature for the individual incubations.

1. Replace GUS staining buffer with 1 ml of 80 % ethanol by
pipetting. Shake gently on an orbital shaker or rocking plate
for 12 h ( see Note 15 ).
2. Change the 80 % ethanol and continue incubation until the
plant material turns yellowish white (usually, a further 12 h
should be suffi cient).
3. Replace the 80 % ethanol by pipetting with 1 ml of 0.25 M
HCl/20 % methanol. Incubate for 15 min at 55 °C and
remove the solution by pipetting ( see Note 16 ).
4. Add 1 ml of 7 % NaOH/60 % ethanol. Incubate for 15 min,
then remove the solution by pipetting ( see Notes 7 and 17 ).
5. Add 1 ml of 40 % ethanol. Incubate for 10 min, then remove
the solution by pipetting.
6. Add 1 ml of 20 % ethanol. Incubate for 10 min, then remove
the solution by pipetting.
7. Add 1 ml of 10 % ethanol. Incubate for 10 min, then remove
the solution by pipetting.
8. Add 1 ml of 5 % ethanol/50 % glycerol. Incubate for 30 min,
then remove the solution by pipetting.
9. Add 1 ml of 50 % glycerol.
10. Store at 4 °C or continue to process as described below
(Subheading 3.4 , see Note 18 ).

3.4 Sample
Preparation

For genetic screen of individual M2 families, use at least 20-30
seedlings per family and per slide ( see Note 19 ).

1. Carefully place the cleared seedlings (or other plant tissues)
onto the microscopic slide and mount them into a thin layer of
50 % glycerol ( see Note 20 ).