Abstract
For immunocytochemistry, cultured bovine corneal endothelial cells (CBCEC) and bovine corneal cryosections were utilized. Preparations were fixed, permeabilized, and incubated with primary rabbit anti-rat aquaporin 1 (AQP1) antibody followed by rhodamine-conjugated secondary antibody, and were counter-stained with Sytox nuclear acid stain. Confocal microscopy of CBCEC in the x, y, and z planes showed rhodamine fluorescence, indicating the presence of AQP1 antibody localized to the apical and basolateral domains of the plasma membrane, but not to the membranes of intracellular compartments or other subcellular locations. Preabsorption with control antigenic peptide yielded no positive staining. Similar results were obtained using freshly dissected bovine corneas; in addition, these images showed AQP1 distributed to the plasma membranes of keratocytes. No AQP1 staining was seen in corneal epithelium, and no staining was observed in CBCEC layers exposed to AQP3, AQP4, and AQP5 antibodies.
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