Telocytes and stem cells in limbus and uvea of mouse eye.

Eyes from four C57BL/6 mice (12 months old) were used for the ultrastructural study after the Institutional Ethical Committee approval. Small samples of about 1 mm 3 were fixed by immersion in 4% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4. Samples were post-fixed in 1% OsO 4 with 1.5% K 4 Fe(CN) 6 (potassium ferrocyanide reduced osmium) in 0.1 M cacodylate buffer. Samples were further dehydrated in increased graded of ethanol followed by propylene oxide and embedded in Epon [ 31 ]. Semi-thin sections (1 μm thick) were stained with 1% toluidine blue and examined by light microscopy (Nikon Eclipse E600, Tokyo, Japan).

Transmission electron microscopy

Transmission electron microscopy

(TEM) was performed on 60 nm thin sections stained with uranyl acetate and lead citrate using a Morgagni 268 electron microscope (FEI Company, Eindhoven, The Netherlands) at 80 kV. Digital electron micrographs were acquired with a MegaView III CCD and iTEM-SIS software (Olympus, Soft Imaging System GmbH, Münster, Germany). All measurements were performed with iTEM-SIS software, using 50 randomly selected structures/images.

Electron microscope tomography

Electron microscope tomography

(ET) was performed on 250-nm thick sections of Epon-embedded tissue [ 32 ] using a Tecnai G2 Spirit BioTwin transmission electron microscope with a single-tilt specimen holder (FEI Company) at 100 kV. Electron tomographic data sets were recorded with a MegaView G2 CCD camera (Olympus) in ET mode. Projection images (1024 × 1024 pixels) were acquired at 1-degree angular increments from −65 to +65 degrees around an axis perpendicular to the optical axis of the microscope, at 36,000× magnification (pixel size 2.65 nm). After data alignment, the data sets were reconstructed into 3D volume (data collection, reconstruction and visualization) using Xplore3D Tomography Suite software (FEI Company). Amira 5.0.1 software (Visage Imaging GmbH, Berlin, Germany) was used for 3D imaging.