Immunocytochemical localization of aquaporin-1 in bovine corneal endothelial cells and keratocytes

For immunocytochemistry, cultured bovine corneal endothelial cells (CBCEC) and bovine corneal cryosections were utilized. Preparations were fixed, per meabilized, and incubated with primary rabbit anti-rat aquaporin 1 (AQP1) a ntibody followed by rhodamine-conjugated secondary antibody, and were count erstained with Sytox nuclear acid stain. Confocal microscopy of CBCEC in th e x, y, and z planes showed rhodamine fluorescence, indicating the presence of AQP1 antibody localized to the epical and basolateral domains of the pl asma membrane, but not to the membranes of intracellular compartments or ot her subcellular locations. Preabsorption with control antigenic peptide yie lded no positive staining. Similar results were obtained using freshly diss ected bovine corneas; in addition, these images showed AQP1 distributed to the plasma membranes of keratocytes. No AQP1 staining was seen in corneal e pithelium, and no staining was observed in CBCEC layers exposed to AQP3, AQ P4, and AQP5 antibodies.