Sigma Duolink In Situ Kit (DUO92101) was used for this assay. Embryos were fixed as for regular antibody staining. After overnight staining with primary antibodies at 15°C, the sample was stained at 37°C for 1 hour with secondary antibodies that have oligonucleotide probes attached. Connector oligos were hybridized to the probes and served as templates for circularization by enzymatic ligation when in close proximity. The ligation reaction was incubated at 37°C for 30 min. The circularized DNA strands were used for rolling circle amplification (RCA), and the RCA product was detected by hybridizing fluorescently labeled oligos. The RCA reaction was incubated at 37°C for 100 min. After each step, slides were washed with tris-buffered saline (TBS) + 0.2% Triton X-100 for 5 min. Slides were mounted in DAPI.

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