objective lens, Nikon LU5 laser bed (488 and 561 nm laser
lines), and Andor DU-897X-5254 EMCCD camera.
5. Capture 15 images (5 phases, 3 angles) with the NIS
Elements v4.1.
6. Reconstruct the images using slice reconstruction in NIS elements followed by post-imaging analysis using NIS Elements
v4.1 (see Note 8). As shown in Fig. 2c, d, distinct from conventional confocal imaging (Fig. 2a, b), deconvolution images
of SIM showed that part of AtSar1c-GFP-labeled ERESs are
separated from Golgi marker mRFP-EMP12 while the rest are
in a close proximity to Golgi rather than colocalized. On the
other hand, almost all ERESs labeled by AtSar1a-GFP are
closely associated with Golgi instead of colocalization observed
by conventional confocal. These results are consistent with
high-resolution imaging such as transmission electron microscopy showing ERESs and Golgi are distinct membrane-bound
organelles [17]. Thus, comparing with conventional confocal,
super-resolution imaging such as SIM analysis may reflect the
bona fide spatial relationship between ERESs and Golgi apparatus. Further, different localizations of these two GFP-fused
Sar1 homologs demonstrated the existence of distinct population of ERESs in plant.
4 Notes
1. The formation of a top protoplast layer is important for the
washing steps. To get a good top protoplast layer in all washing
steps, it is important that no brake is used for the centrifugation
with the swinging bucket rotor.
2. If the protoplasts cannot float up to the top with proper centrifugation conditions, check the morphology and size of the
protoplasts. Small protoplast with tiny vacuole may not float
up to the top due to the density. Using higher concentration of
sucrose in the electroporation buffer may enable the floating of
protoplasts to the top.
3. Using the Pasteur pipette tip push aside the protoplast and
make a hole on the top protoplast layer. Then poke the Pasteur
pipette through the top layer via the hole carefully. If not, the
Pasteur pipette will drag down some protoplasts to the underlying layer and cause the loss of protoplast cells.
4. The plant protoplasts are fragile, operate gently when
transferring them.
5. The quality of the plasmid DNA is crucial. The protoplasts will
die after transfection if the plasmid is not pure enough. In
Protoplasts Isolation and Transient Expression
163
lines), and Andor DU-897X-5254 EMCCD camera.
5. Capture 15 images (5 phases, 3 angles) with the NIS
Elements v4.1.
6. Reconstruct the images using slice reconstruction in NIS elements followed by post-imaging analysis using NIS Elements
v4.1 (see Note 8). As shown in Fig. 2c, d, distinct from conventional confocal imaging (Fig. 2a, b), deconvolution images
of SIM showed that part of AtSar1c-GFP-labeled ERESs are
separated from Golgi marker mRFP-EMP12 while the rest are
in a close proximity to Golgi rather than colocalized. On the
other hand, almost all ERESs labeled by AtSar1a-GFP are
closely associated with Golgi instead of colocalization observed
by conventional confocal. These results are consistent with
high-resolution imaging such as transmission electron microscopy showing ERESs and Golgi are distinct membrane-bound
organelles [17]. Thus, comparing with conventional confocal,
super-resolution imaging such as SIM analysis may reflect the
bona fide spatial relationship between ERESs and Golgi apparatus. Further, different localizations of these two GFP-fused
Sar1 homologs demonstrated the existence of distinct population of ERESs in plant.
4 Notes
1. The formation of a top protoplast layer is important for the
washing steps. To get a good top protoplast layer in all washing
steps, it is important that no brake is used for the centrifugation
with the swinging bucket rotor.
2. If the protoplasts cannot float up to the top with proper centrifugation conditions, check the morphology and size of the
protoplasts. Small protoplast with tiny vacuole may not float
up to the top due to the density. Using higher concentration of
sucrose in the electroporation buffer may enable the floating of
protoplasts to the top.
3. Using the Pasteur pipette tip push aside the protoplast and
make a hole on the top protoplast layer. Then poke the Pasteur
pipette through the top layer via the hole carefully. If not, the
Pasteur pipette will drag down some protoplasts to the underlying layer and cause the loss of protoplast cells.
4. The plant protoplasts are fragile, operate gently when
transferring them.
5. The quality of the plasmid DNA is crucial. The protoplasts will
die after transfection if the plasmid is not pure enough. In
Protoplasts Isolation and Transient Expression
163