2. Transfer aggregates from PCR tube or culture dish to fixative
using an end-cut 200P- tip fitted onto a 200P manual pipettor.
3. Fix the aggregate for 15 min at room temperature.
4. Wash 3 times for 15 min each with PBST (PBS + 0.1% Triton
X). If using Dent’s fixative the embryos must be rehydrated
through a series MeOH, dH 2 O, and PBST.
5. Block nonspecific antibody binding with 10% goat serum in
PBST for 1 h at room temperature.
6. Incubate with first antibody at 4
C for overnight.
7. Wash three times for 15 min each with PBST.
8. Incubate with 2
antibody at 4
C for overnight.
9. Wash three times for 15 min each with PBST.
10. (optional) Stained aggregates can be cleared to image inside of
the aggregates by following series of dehydration, soaking in
Murray’s clear until visibly clear under a stereoscope (~5 min).
11. Transfer to a glass-bottomed imaging chamber for high resolution confocal imaging.
To capture the progression of mesenchymal-to-epithelial
transition, live aggregates can be imaged using a confocal
microscope by following steps 12–16 (Fig. 2).
12. Microinject mRNA for protein of interest at one or two cell
stage of embryo. For example, injecting mRNA encoding
GFP-ZO-1 proteins can reveal progressive changes as cells
transition from mesenchymal-to-epithelial phenotypes. To
microinject, load the pulled glass capillary with desired
mRNA (~3 μl) using a 10P pipettor fitted with a microloader
pipette tip. The opening size of injecting glass needle should be
minimized to prevent inducing an open hole in embryo during
injection and can be adjusted by clipping the pulled end of glass
capillary with sharp forceps. To quantify the amount of mRNA,
inject small volumes into a drop or dish of mineral oil. The
volume of the injected bolus can be measured with a stereomicroscope equipped with a graduated reticle scale from the
diameter of the drop of mRNA in mineral oil. To minimize
the damage and aid healing after injection, place the embryo in
3% Ficoll/1Â MBS during microinjection. Even expression
within deep cells can be obtained by injecting 3 to 4 sites across
the animal pole of the 1-cell stage embryo (see Note 6).
13. Just prior to ectoderm dissection, screen and select embryos
with optimal expression of fluorescently labeled proteins under
the fluorescent stereomicroscope (see Note 7).
14. Assemble aggregates following the methods above (see Subheadings 3.1 and 3.2).
Xenopus Deep Cell Aggregates: A 3D Model for MET
281
using an end-cut 200P- tip fitted onto a 200P manual pipettor.
3. Fix the aggregate for 15 min at room temperature.
4. Wash 3 times for 15 min each with PBST (PBS + 0.1% Triton
X). If using Dent’s fixative the embryos must be rehydrated
through a series MeOH, dH 2 O, and PBST.
5. Block nonspecific antibody binding with 10% goat serum in
PBST for 1 h at room temperature.
6. Incubate with first antibody at 4
C for overnight.
7. Wash three times for 15 min each with PBST.
8. Incubate with 2
antibody at 4
C for overnight.
9. Wash three times for 15 min each with PBST.
10. (optional) Stained aggregates can be cleared to image inside of
the aggregates by following series of dehydration, soaking in
Murray’s clear until visibly clear under a stereoscope (~5 min).
11. Transfer to a glass-bottomed imaging chamber for high resolution confocal imaging.
To capture the progression of mesenchymal-to-epithelial
transition, live aggregates can be imaged using a confocal
microscope by following steps 12–16 (Fig. 2).
12. Microinject mRNA for protein of interest at one or two cell
stage of embryo. For example, injecting mRNA encoding
GFP-ZO-1 proteins can reveal progressive changes as cells
transition from mesenchymal-to-epithelial phenotypes. To
microinject, load the pulled glass capillary with desired
mRNA (~3 μl) using a 10P pipettor fitted with a microloader
pipette tip. The opening size of injecting glass needle should be
minimized to prevent inducing an open hole in embryo during
injection and can be adjusted by clipping the pulled end of glass
capillary with sharp forceps. To quantify the amount of mRNA,
inject small volumes into a drop or dish of mineral oil. The
volume of the injected bolus can be measured with a stereomicroscope equipped with a graduated reticle scale from the
diameter of the drop of mRNA in mineral oil. To minimize
the damage and aid healing after injection, place the embryo in
3% Ficoll/1Â MBS during microinjection. Even expression
within deep cells can be obtained by injecting 3 to 4 sites across
the animal pole of the 1-cell stage embryo (see Note 6).
13. Just prior to ectoderm dissection, screen and select embryos
with optimal expression of fluorescently labeled proteins under
the fluorescent stereomicroscope (see Note 7).
14. Assemble aggregates following the methods above (see Subheadings 3.1 and 3.2).
Xenopus Deep Cell Aggregates: A 3D Model for MET
281