Andor spinning disc confocal microscope

Sorted populations of SCC131 were grown on cover-slips overnight and then fixed with chilled aceto-methanol (1:1). 0.03% Saponin (Calbiochem, Germany) was used for permeabilization followed by blocking with 3% BSA. Rabbit monoclonal antibody against β-catenin and mouse monoclonal antibody against CD24, CD44 (Cell signaling technology) were added at a dilution of 1:200 and incubated overnight. It was then probed with anti-rabbit-FITC and anti-mouse Alexa-Flour 633nm conjugated secondary antibody (molecular probes) and counter stained with DAPI (Invitrogen) for nuclear staining. Images were taken under a confocal microscope (Andor Spinning Disc Confocal Microscope, Andor Technology, Belfast, Ireland) at 60X magnification.

Confocal images of DRGs and Neu7 cells on eight-well chamber slides were captured using a 20X lens on an Andor spinning disc confocal microscope (Andor Technology Ltd., Bristol, UK). To examine the immunopositive cells, confocal z-stack images were captured at 1 µm z step distance apart. Spinal cord slices were also imaged using the Andor spinning disc confocal microscope at 10×. Projected confocal image stacks were captured of immunohistochemically stained spinal cord slices. All imaging was carried out using the same exposure time and emission gain for all spinal slices.

The human breast cancer cell lines were applied for the visualization of cellular uptake of nanolipid vesicles by confocal laser scanning microscopy. The human breast cancer cell lines MCF-7 cells (estrogen receptor-positive) and MDA-MB-453 cells (estrogen receptor-negative) were cultivated for 24 h on the top of coverslips in six-well culture plates (3 mL/well at a density of 10⁴ cells/mL). The cell lines MCF-7 and MDA-MB-453 were obtained from the Chittaranjan Cancer Research Institute, Kolkata and the Indian Institute of Chemical Biology, Kolkata, respectively. Antibody conjugated nanolipid vesicles were then applied at a concentration range of 50 μg/mL and 100 µg/mL in the MCF-7 and MDA-MB-453 cell culture medium. Cells were co-incubated with FITC loaded nanolipid vesicles without the drug as an additional control. Both the cancer cell lines were washed thoroughly for three hours of incubation and then fixed with a paraformaldehyde aqueous solution of 4% (v/v). After fixing both cell lines for 15 min, they were rinsed with phosphate buffer saline (pH 7.4). For the next step, the coverslips were placed carefully so that both the cell lines adhered to the coverslips upon the slide. The slides were dried and kept under the confocal laser scanning microscopy (Andor Spinning Disc Confocal Microscope, Andor Technology, Ireland, UK) [29 (link)].