Fig. 4. Identification of NHE3-expressing cells as PNA⁺ MRCs in gill sections from adult rainbow trout. (A) NHE3 protein was detected in gill (G) and kidney (K) protein extracts using Western blots (N=6) to reveal a 98 kDa immunoreactive band (lanes 1 and 2) that was absent after the NHE3 antibody was incubated with excess peptide against which the antibody was raised (lanes 3 and 4). Size markers are given in kDa. (B) Co-localization of NHE3 (green) and Na⁺/K⁺-ATPase (red) in MRCs (nuclei are stained blue); NHE3-expressing MRCs are labelled with arrows and NHE3 negative MRCs are indicated by stars. (C) Localization of NHE3 to PNA⁺ MRCs; red indicates NHE3, green indicates PNA and nuclei are stained blue. (D) The NHE3 immunofluorescence disappeared with omission of primary NHE3 antibody. Scale bar in A, 40 µm; and in C, 10 µm.