High-proof Ethanol Fixation of Larval and Juvenile Fishes for Clearing and Double Staining
The fixation of larval and juvenile fishes in 95% ethanol can be substituted for formalin and bears several advantages for morphological and molecular studies: 1) specimens clear and double stain rapidly and brilliantly; 2) otoliths are preserved; and 3) high-quality DNA is available from the tissues. We present merits and limitations of 70% ethanol and 95% ethanol as alternative fixatives to 4% buffered formalin. In particular, we compare our results of clearing and double staining teleost larvae and juveniles from these three fixatives and those that have been frozen at −20°C prior to the initial fixation. With our results, we can refute the long-standing notion that ethanol-fixed specimens disintegrate during clearing and staining.
(A) Platichthys flesus, MNHN 2014 2984, 22.0 mm SL. (B) Head and (C) part of the vertebral column of a Syngnathidae sp., MNHN 2014 2980, 42.0 mm SL. (D) Skeletal elements of Pomatoschistus minutus, MNHN 2014 2995, 22.0 mm SL. All specimens were initially dry frozen, thawed in water, and subsequently placed for three days into 95% ethanol before they were cleared and double stained.
Alosa aestivalis. Cleared and double stained juveniles in comparison. (A) MNHN 2014 2997, 42.8 mm SL, fixed in 70% ethanol, (B) MNHN 2014 2998, 40.4 mm SL, fixed in 95% ethanol, (C) MNHN 2014 2999, 45.3 mm SL, fixed in 4% buffered formalin, and (D) VIMS 3303, 46.5 mm SL, frozen at −20°C with a following fixation in 95% ethanol. Scale bars = 5 mm.
Alosa aestivalis. Skeletal elements of cleared and double stained juveniles fixed in 70% ethanol (MNHN 2014 2997, 42.8 mm SL), 95% ethanol (MNHN 2014 2998, 40.4 mm SL), and 4% buffered formalin (MNHN 2014 2999, 45.3 mm SL) in comparison. (A–C) Oral jaws, suspensorium, and opercular bones; (D–F) dorsal gill arches; (G–I) ventral gill arches; (J–L) caudal fin; (M–O) dorsal fin. Scale bars = 1 mm.
Contributor Notes
Associate Editor: W. L. Smith.