Download BOX 2

BOX 2
Limitations of fluorescent dyes and primary antibody species can be partially superseded when
using validated and optimized reagents with established expression patterns. We provide the
following advice to maximize the information collected from each embryo:
1. Two targets with non-overlapping subcellular localization (e.g. nuclear GATA6 and cytoskeletal
Phalloidin) can be stained simultaneously in the same channel. A DAPI mask can be used to
generate separate “virtual” nuclear and non-nuclear channels. It is important to independently
confirm the subcellular distribution of each antigen at an optimized primary antibody dilution first.
2. A second target can be detected using serial labeling and imaging with a primary antibody
raised in the same species as the primary antibody already used to detect a first target (e.g.,
mouse anti-CDX2 and mouse anti-GATA3). It is important to image the first stain before
proceeding to the second one. Serial staining is most informative when the second target has a
greater distribution or higher signal to noise compared to the first target, and it is ideal when the
two targets are in non-overlapping subcellular compartments (e.g. GATA6 and CK7). Fc/Fab
blocking after the first stain and use of Fab secondaries can considerably reduce, but not
completely eliminate, serial-staining crosstalk. Refer to the Jackson ImmunoResearch website for
an example of a serial staining protocol, keeping in mind that incubation timing will need to be
adjusted to match what described in Part 3. Relevant controls should be performed, crosstalk
measured empirically with sequential imaging, and results interpreted carefully.