2.5| Northern blotting
Northern blotting was performed, as previously described (Yamaguchi et al., 2000). Briefly, using cDNA fragments for genes encoding NKA α subunit, CAc, NHE, and NKCC as templates, digoxigenin-labeled RNA probes were synthesized according to the manufacturer’s instructions for the 10x DIG RNA labeling mix (Roche, Basel, Switzerland). Total RNA was extracted from the most posterior pair of gills using Isogen reagent (Nippon Gene), and 8.5 µg of total RNA was electrophoresed in 1% agarose gel containing 1x 3-(N -morpholino)propanesulfonic acid (MOPS) and 5.5% formalin. Next, RNA was transferred from the gel to a positively charged nylon membrane (Roche) overnight. The resultant membrane was prehybridized using a solution containing 5x saline-sodium citrate (SSC), 50% formamide, 5x Denhardt’s solution, 0.5% sodium dodecyl sulfate (SDS), and 0.005% transfer RNA (tRNA) and then hybridized with digoxigenin-labeled RNA probes overnight. Finally, the membrane was washed, blocked, and treated with alkaline phosphatase–conjugated anti-digoxigenin antibody (Roche) at a dilution of 1:2000. Signals were detected using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate. The intensity of detected signals of Northern blotting was normalized with amount of electrophoresed ribosomal RNA, both of which were quantified using image processing program, ImageJ (National Institute of Health, Maryland, USA). The relative expression level was calculated as the expression in 513.3 mmol/L NaCl+MCK (Fig. 5) or 4.3 mmol/N NaCl solution (Fig. 9) was 1.0.