Academic Language Program
Method
A. Sample staining
To start the sample staining, 15-20 seedlings were added into each well containing 2 ml of Fluorol
Yellow 088 (FY088). The lid of the plate was closed and sealed with parafilm. The plate was placed in
a water bath on an aluminium foil raft at 70 °C for 20 min. Next, the seedlings were transferred from
the FY088 solution to a new 12-well plate containing 2 ml water and were incubated for 1 min at
room temperature. Water was removed by using a pipette and 2 ml of 0.5% aniline blue solution was
added to each sample. This solution was incubated for 20 min in the dark at room temperature.
After incubation in aniline blue, the seedlings were moved to new 12-, and 24-well plates containing
2-3 ml water and were incubated for 10 minutes in the dark.
B. Sample preparation
For sample preparation, two strips of parafilm were cut and wrapped 2-3 times around both ends of
the microscope slide. After wrapping both ends, a few drops of water were added in the centre of
the slide between two parafilm strips. Using tweezers, the seedlings were positioned onto this liquid
and a cover glass slide was used to protect the sample.
C. Sample imaging
For the third part of this experiment, sample imaging, an inverted confocal microscope was
prepared for use; lasers (for GFP: 488 nm excitation, emission 500-550 nm) and objectives were set.
For a broad overview of suberin localization in the root, performing tile scan imaging using either a
10× or 20× objective (in dry or water-immersion mode) is recommended. For higher resolution
images, using a 40× or 63× objective immersed in water or oil is recommended.
A glass slide or chamber with seedlings was mounted and placed on the stage of the confocal
microscope. The position list was activated, and tile scanned (25 × 34 tiles) with 20% overlap
between images. Next, the root closest to the central point of the scanning area was found and the
position was marked. The position was deactivated, and scanning was started. Lastly, confocal
images were exported in TIFF or JPEG format. Images were opened in ImageJ or other software and
were analysed.
After staining, preparation and imaging, data analysis was performed using ImageJ. A line along the
root was drawn in ImageJ, after which the root length was measured. The procedure was repeated
in order to measure the length of the suberized region of the root. For further processing of the
data, Excel was used.
Further in the process, seeds were sterilized with ethanol and transferred to 2 ml Eppendorf tubes.
Next, 1 ml of 70% EtOH was added, put aside for 10 minutes, and left to dry under the clean bench,
respectively.
For the last part, 10 g sucrose was combined with 2.3 g MS salts and 0.5 g MES. The pH was adjusted
to 5.9 with KOH. 8 g agar was added and this was filled with ddH2O until 1 L was reached. The
medium was autoclaved for 20-30 minutes and allowed to cool to 55 °C. To make the 0.003% Fluorol
Yellow 088 solution, we made Yellow 088 in lactic acid at room temperature before each
experiment. Moreover, a 50 ml Falcon tube was attached to a vortex mixer with adhesive tape and
vortex for 10-15 min. Finally, a 0.5% Aniline blue solution was prepared by adding Aniline blue in
Milli-Q Water and storing it in a dark place at room temperature.
Method
A. Sample staining
To start the sample staining, 15-20 seedlings were added into each well containing 2 ml of Fluorol
Yellow 088 (FY088). The lid of the plate was closed and sealed with parafilm. The plate was placed in
a water bath on an aluminium foil raft at 70 °C for 20 min. Next, the seedlings were transferred from
the FY088 solution to a new 12-well plate containing 2 ml water and were incubated for 1 min at
room temperature. Water was removed by using a pipette and 2 ml of 0.5% aniline blue solution was
added to each sample. This solution was incubated for 20 min in the dark at room temperature.
After incubation in aniline blue, the seedlings were moved to new 12-, and 24-well plates containing
2-3 ml water and were incubated for 10 minutes in the dark.
B. Sample preparation
For sample preparation, two strips of parafilm were cut and wrapped 2-3 times around both ends of
the microscope slide. After wrapping both ends, a few drops of water were added in the centre of
the slide between two parafilm strips. Using tweezers, the seedlings were positioned onto this liquid
and a cover glass slide was used to protect the sample.
C. Sample imaging
For the third part of this experiment, sample imaging, an inverted confocal microscope was
prepared for use; lasers (for GFP: 488 nm excitation, emission 500-550 nm) and objectives were set.
For a broad overview of suberin localization in the root, performing tile scan imaging using either a
10× or 20× objective (in dry or water-immersion mode) is recommended. For higher resolution
images, using a 40× or 63× objective immersed in water or oil is recommended.
A glass slide or chamber with seedlings was mounted and placed on the stage of the confocal
microscope. The position list was activated, and tile scanned (25 × 34 tiles) with 20% overlap
between images. Next, the root closest to the central point of the scanning area was found and the
position was marked. The position was deactivated, and scanning was started. Lastly, confocal
images were exported in TIFF or JPEG format. Images were opened in ImageJ or other software and
were analysed.
After staining, preparation and imaging, data analysis was performed using ImageJ. A line along the
root was drawn in ImageJ, after which the root length was measured. The procedure was repeated
in order to measure the length of the suberized region of the root. For further processing of the
data, Excel was used.
Further in the process, seeds were sterilized with ethanol and transferred to 2 ml Eppendorf tubes.
Next, 1 ml of 70% EtOH was added, put aside for 10 minutes, and left to dry under the clean bench,
respectively.
For the last part, 10 g sucrose was combined with 2.3 g MS salts and 0.5 g MES. The pH was adjusted
to 5.9 with KOH. 8 g agar was added and this was filled with ddH2O until 1 L was reached. The
medium was autoclaved for 20-30 minutes and allowed to cool to 55 °C. To make the 0.003% Fluorol
Yellow 088 solution, we made Yellow 088 in lactic acid at room temperature before each
experiment. Moreover, a 50 ml Falcon tube was attached to a vortex mixer with adhesive tape and
vortex for 10-15 min. Finally, a 0.5% Aniline blue solution was prepared by adding Aniline blue in
Milli-Q Water and storing it in a dark place at room temperature.
