Imager z2 microscope

The two-layer hTECs were produced following the self-assembly approach as described previously [31 (link),35 (link),36 (link)]. Briefly, corneal fibroblasts were cultured in the presence of ascorbic acid (50 μg/mL, Sigma) for 35 days to promote the production of their own extracellular matrix. Two fibroblast sheets were then superimposed to form a reconstructed stroma, on which hLECs (P2) were seeded. Reconstructed tissues were cultured for 7 days under submerged conditions in complete DHc supplemented with ascorbate and then transferred to the air–liquid interface for 7 days in EGF-free DHc to induce epithelial differentiation. Reconstructed partial thickness corneas were then wounded using an 8 mm biopsy punch. After wounding, hTECs were placed over two additional fibroblast sheets to allow re-epithelialization over a natural extracellular matrix and culture was continued at the air–liquid interface. Wound closure was monitored macroscopically for 8 days and photographed at 24 h intervals with a Zeiss Imager Z2 microscope (Zeiss Imager.Z2 microscope (Zeiss Canada Ltd.). All experiments were conducted in quadruplicate (n = 4). The wound surface over time was measured using ImageJ software (Wayne Rasband).

XMP13 probe (D-1413, Metasystems) specific for mouse Chromosome 13 was used for chromosome identification. FISH on chromosome spread was performed according to manufacturer’s instructions. The probe was added and coverslip placed, sealed with rubber cement, denatured by heating slide at 75°C for 2 min, and incubated in humidified chamber at 37°C overnight. Slides were washed and stained with 0.5 μg/ml DAPI in VectaShield antifade medium. Digital images were captured using a CCD camera on a Zeiss Imager Z2 microscope. The coordinates of the chromosome were recorded with the venire scale along the top and side of the microscope stage. The slides were washed and performed with telomere FISH as described above. After staining with DAPI again, fluorescence from chromosomes and telomeres was digitally imaged using the same microscope according to the recorded coordinates. The telomeres of chromosome 13 were revealed by comparison of the images from the same coordinates.

Cells (1 μl) from the doxorubicin uptake assay were placed into the wells of multitest slide 15 (MP Biomedicals) that were pre-coated with 1 mg/ml concanavalin A (MP Biomedicals). The slides were air dried completely and 1 μl of mounting medium with DAPI (UltraCruz) was added to each well. A cover glass was sealed onto the slide, images were taken with an epifluorescent microscope (Olympus B53 upright epifluorescent microscope equipped with an Olympus XM10 camera at 63X with Texas red filter to detect DOX or with the Zeiss Imager Z2 microscope equipped with Zeiss AxioCam MRC camera) and processed with Image J.