Isopentyl acetate

For scanning electron microscopy, leech specimens were treated with 16% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) or relaxation solution (4.8 mM NaCl₂, 1.2 mM KCl, 10 mM MgCl₂, 8% EtOH) while feeding or relaxing. After treatment, the head region containing the proboscis was cut and fixed in 4% PFA at room temperature overnight. The tissues were washed three times with PBT (1X PBS + 0.1% Tween-20) for 20 min at room temperature, and then fixed in 1% osmium tetroxide (Ted Pella Inc., Redding, CA, USA) in 1 M PBS for 1 h. Osmium tetroxide was removed by washing three times with PBT. Thereafter, the tissues were gradually dehydrated with ethanol (30%, 50%, 60%, 70%, 80%, 90%, 95%, 100% in 1X PBS) for 20 min per step. Dehydrated tissues were treated with stepwise concentrated isopentyl acetate (Alfa Aesar, Ward Hill, MA, USA) (isopentyl acetate: EtOH = 1:3, 1:1, and 3:1) for 15 min per step, and then transferred to 100% isopentyl acetate. After the solution was removed, the samples were dried for 3 days in a fume hood. Dried samples were coated with gold particles and examined with an UltraPlus field emission scanning electron microscope (Carl Zeiss, Oberkochen, BW, Germany).