Transparent Visualization of Fish Larvae

Fish larvae were bleached and transparentized according to previous reports^{51 (link),52 (link)}, with several modifications. Formalin (10% formalin solution), potassium hydroxide (KOH), hydrogen peroxide, Triton-X, phosphate-buffered saline (PBS), HEPES, and glycerin were purchased from Wako Pure Chemical Industries Ltd. (Tokyo, Japan). All other chemicals used in this study were of analytical grade. O. javanicus larvae (exposure group n = 6; negative control n = 2) were fixed in 10% formalin at 4 °C for 3 days. After removal of formalin, larvae were incubated in pre-bleaching solution (0.3% hydrogen peroxide in PBS) at room temperature overnight. Pre-bleaching solution was replaced with bleaching solution (3% hydrogen peroxide in PBS) and incubated at room temperature overnight. After bleaching, the bleaching solution was replaced with a tissue transparency solution (5% formalin, 5% Triton X-100, 1% KOH in PBS) and incubated at 42 °C for 24 h. In O. latipes, which has a less pigmented peritoneum than O. javanicus, bleaching was omitted, i.e., the tissue transparency solution was directly added after fixation, and incubated at 42 °C for 48 h. Transparency of each sample was confirmed under the microscope.