Histological Characterization of Lens Capsule

Immediately after samples were removed from culture, they were placed in 4% paraformaldehyde for fixation for at least one week and then embedded in paraffin. Once all samples were in paraffin, they were sectioned along the sagittal plane and stained on the same day. Tissue were stained with hematoxylin and eosin for morphological analysis, α-smooth muscle actin α-SMA (clone 14A, Cell Marque) for myofibroblast formation and intercapsular adhesion^{28 (link)–30 (link)}, Ki-67 (clone 30–9, Ventana) for proliferation^{31 (link)} and vimentin (clone V9, Ventana) a marker of both undifferentiated lens epithelium as well as differentiating fiber cells^{32 (link)}. All immunostainings were done with the BenchMark® ULTRA device (Ventana Medical Systems, Inc.). We also included negative immunostain controls to support the validity of our stains and identify possible experimental artefacts or background noise. These controls were processed in the same manner with the automated staining device but without the primary antibodies. Capsular adhesion, cellular morphology and the degree of staining of α-SMA, Ki-67 and vimentin were studied at the equatorial region of the capsule and at the center of the posterior capsule.