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.