Double immunohistochemical staining of eel gill sections, (A-D) Na+/K+-ATPase α-subunits (Naα5) and osmotic stress transcriptional factor 1 (Ostf1), (E-H) Ostf1 and cystic fibrosis transmembrane regulator (Cftr), (I-L) Naα5 and phosphorylated extracellular signal regulated kinase (p-ERK) prepared from seawater acclimating (day1, day 3, and day 7) Japanese eels ( Anguilla japonica ). The figures represented the general trends of four independent experiments. (A-H) Eel Ostf1 expressed in most cytoplasm of MRCs but not at the apical pit after day 3 freshwater to seawater transfer. (A-D) Double staining of Naα5 and Ostf1. Red color indicated the MRCs, which were positively stained by Naα5 antibody. Increase of MRCs in primary filament region was observed after the transfer. Additionally, Ostf1 staining was marked by green color, which expressed and co-localized with MRCs (yellow) from day 3 after fresh water to seawater transfer. (E-H) Double-staining of Cftr and Ostf1. Red color marked Cftr, which started its expression at apical pit of MRCs from day 3 after fresh water to seawater transfer. The Ostf1 expressed cells were recognized by green color, which did not overlap with Cftr staining at the apical pits of MRCs. Clear red color at the apical pits was observed. (I-L)Induction of p-ERK expressed in gill cells after day 3 of fresh water to seawater transfer. Double-staining of Naα5 and p-ERK. Red color marked MRCs. Green color indicated p-ERK positively stained cells, which started its expression at day 3 after fresh water to seawater transfer. It should be noted that only some of the MRCs showed ERK phosphorylation (yellow) at day 3, while all MRCs showed ERK phosphorylated at day 7 after fresh water to seawater transfer. (M) Magnified image of Ostf1 in MRCs . It clearly showed that the Ostf1 (green) did not co-localized at the apical pits with Cftr (red). (N) Magnified image of ERK phosphorylated MRCs. At day 7 after fresh water to seawater transfer, p-ERK expressed in MRCs. Scale bars = 100 μm (A-D), 80 μm (E-L), and 50 μm (M-N).