Fig. 6. Classification of mitochondria-rich cells (MRCs) in the yolk-sac membrane of Mozambique tilapia embryos into four types by means of quintuple-color immunofluorescence staining: (A) type-I MRC (at 72h of seawater-to-freshwater transfer), (B) type-II MRC (at 72h of seawater-to-freshwater transfer), (C) type-III MRC (at 72h of seawater-to-freshwater transfer) and (D) type-IV MRC (at 72h of freshwater-to-seawater transfer). The immunofluorescence signals for Na⁺/K⁺-ATPase (red), NKCC1a (blue), NCC (cyan), NHE3 (yellow) and CFTR (green) are shown as separate channels, and the five channels are merged in Merged X–Y and X–Z planes. Merged X–Z plane, the X–Z optical section cut transversely at the horizontal lines indicated in Merged X–Y plane. Merged X–Y plane, the X–Y optical section cut at the lines indicated in Merged X–Z plane. n, nucleus; ac, accessory cell. Scale bar, 10 µm. Schematic diagrams of each of the four cell types are presented in the bottom row, showing the apical or basolateral localization patterns of Na⁺/K⁺-ATPase (red), NKCC1a (blue), NCC (cyan), NHE3 (yellow) and CFTR (green).