Improved fixation of the whole bodies of fish by a double-fixation method with formalin solution and Bouin's fluid or Davidson's fluid

To prevent fixation defects or artifacts in the whole bodies of fish caused by conventional fixatives, such as formalin solution, Bouin's fluid (BF), and Davidson's fluid (DF), the optimal fixatives and fixing method were examined. An improved method of fixing the whole bodies of fish was examined that makes use of a combination of 20% formalin and BF or DF. The fixatives were examined with four representative tissues, i.e., the gill, liver, intestinal tract, and kidney, to evaluate end points including the appearance of degraded tissues and artifacts caused by each fixative, overall morphological clarity of nuclei, staining intensity, and integrity of the other tissues. The best results were obtained when the fresh whole bodies were initially fixed in 20% formalin (primary fixation) at 4 degrees C for 1 h and subsequently fixed in BF for 5 h at 4 degrees C (secondary fixation). Therefore, the current findings led the authors to conclude that the combination of primary fixation with 20% formalin at 4 degrees C for 1 h and secondary fixation with BF at 4 degrees C for 5 h was suitable for fixation of the whole bodies of fish.