FMRFamide patterning over the course of embryonic life has been recorded through immunofluorescence. Embryos at 0 to 144 hours (6 days) post cleavage were extracted from the egg mass and fixed in 4% PFA for 2 hrs at room temperature.  Embryos older than 4 days were pre-treated with 0.5% ethylenediaminetetraacetic acid (EDTA) solution in PBS for 5 minutes to improve primary antibody penetration, and then fixed in 4% PFA for 2 hours at room temperature. Embryos were then washed overnight in 4% Triton X-100 in PBS and incubated in blocking solution, 1% normal goal serum in PBS, overnight at 4 °C.
After blocking overnight, embryos were treated with a primary antibody solution at 1:2000 (in NGS/PBS). Secondary antibody tagged with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (polyclonal) was incubated at 1:20, overnight at 4 °C. Embryos were then mounted on a slide and viewed with fluorescent microscopy.