3. During isolation, place the isolated ectoderm apical side up in
calcium-magnesium free DFA and monitor under a stereoscope to identify the right time to separate the superficial
layer. Tissues cultured too long in CMF-DFA dissociate
completely making it impossible to separate superficial from
deep ectoderm cells.
4. To transfer aggregates we recommend a manual pipettor with a
wide tip opening. We typically cut the end of a 200P tip with
sterilized scissors to enlarge the opening. This allows gentle
transfer aggregates with minimal transient shear stress. Furthermore, the tip reduces the amount of media transferred to
the next chamber or dish.
5. Certain epitopes are sensitive to the type of fixative and duration of the fix. For most studies of the surface layer cells we fix
aggregates using 4% fresh paraformaldehyde in PBS for 15 min
at room temperature. Longer fixation may be required to
preserve deep cells. Some epitopes, notably those found in
membrane bound adhesion proteins like cdh1 or cdh3 (E- or
C-cadherin in the older cadherin nomenclature) or tightjunction protein TJP1, also known as ZO-1. For these cases
we fix aggregates in ice-cold Dent’s fixative (80:20 anhydrous
methanol (aMeOH) to dimethyl sulfoxide (DMSO) [44] overnight at À20
C. Often we fix and stain for F-actin to monitor
cell phenotypes using 4% fresh paraformaldehyde supplemented with 0.2% glutaraldehyde for 15 min at room temperature
and stained with phallacidin or phalloidin. Phalloidin/phallacidin staining for F-actin is not compatible with Methanol. In
order to combine F-actin staining with epitopes requiring
Dent’s fixative we substitute Isopropanol for Methanol. Anhydrous isopropanol must also be used with the dehydration steps
in order to preserve phalloidin/phallacidin binding. If
co-staining for F-actin, we use complementary fluorescent derivatives of phallacidin or phalloidin (1:800) together with the
secondary antibody.
6. Timing is critical to ensure even distribution of injected
mRNA. Best results are obtained by restricting mRNA injections to the first 60 min after fertilization. Injections within
15 min of the first cell division, or later stages often result in
uneven fluorescence signal.
7. We warn against selecting the “brightest embryos” during
screening injected embryos with a fluorescence stereoscope.
High levels of overexpression are not needed for imaging in
the more sensitive confocal microscope and may interfere with
normal protein dynamics or MET. You must use careful titration and expression studies in whole embryos to find expression levels that do not interfere with normal development.
These preliminary studies can validate levels of expression for
live-aggregate studies.
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Hye Young Kim and Lance A. Davidson
calcium-magnesium free DFA and monitor under a stereoscope to identify the right time to separate the superficial
layer. Tissues cultured too long in CMF-DFA dissociate
completely making it impossible to separate superficial from
deep ectoderm cells.
4. To transfer aggregates we recommend a manual pipettor with a
wide tip opening. We typically cut the end of a 200P tip with
sterilized scissors to enlarge the opening. This allows gentle
transfer aggregates with minimal transient shear stress. Furthermore, the tip reduces the amount of media transferred to
the next chamber or dish.
5. Certain epitopes are sensitive to the type of fixative and duration of the fix. For most studies of the surface layer cells we fix
aggregates using 4% fresh paraformaldehyde in PBS for 15 min
at room temperature. Longer fixation may be required to
preserve deep cells. Some epitopes, notably those found in
membrane bound adhesion proteins like cdh1 or cdh3 (E- or
C-cadherin in the older cadherin nomenclature) or tightjunction protein TJP1, also known as ZO-1. For these cases
we fix aggregates in ice-cold Dent’s fixative (80:20 anhydrous
methanol (aMeOH) to dimethyl sulfoxide (DMSO) [44] overnight at À20
C. Often we fix and stain for F-actin to monitor
cell phenotypes using 4% fresh paraformaldehyde supplemented with 0.2% glutaraldehyde for 15 min at room temperature
and stained with phallacidin or phalloidin. Phalloidin/phallacidin staining for F-actin is not compatible with Methanol. In
order to combine F-actin staining with epitopes requiring
Dent’s fixative we substitute Isopropanol for Methanol. Anhydrous isopropanol must also be used with the dehydration steps
in order to preserve phalloidin/phallacidin binding. If
co-staining for F-actin, we use complementary fluorescent derivatives of phallacidin or phalloidin (1:800) together with the
secondary antibody.
6. Timing is critical to ensure even distribution of injected
mRNA. Best results are obtained by restricting mRNA injections to the first 60 min after fertilization. Injections within
15 min of the first cell division, or later stages often result in
uneven fluorescence signal.
7. We warn against selecting the “brightest embryos” during
screening injected embryos with a fluorescence stereoscope.
High levels of overexpression are not needed for imaging in
the more sensitive confocal microscope and may interfere with
normal protein dynamics or MET. You must use careful titration and expression studies in whole embryos to find expression levels that do not interfere with normal development.
These preliminary studies can validate levels of expression for
live-aggregate studies.
284
Hye Young Kim and Lance A. Davidson