Cells were grown on coverslips inserted in six-well plates to attach overnight in the presence of siRBBP6 and pRBBP6 and then treated with the desired concentration of camptothecin (0.25 µM) or GABA (100 µM). The cells were then allowed to incubate for further 24 hours. For apoptosis detection, the cells were washed with cold PBS, and stained with annexin V-FITC/4′,6-diamidino-2-phenylindole (DAPI) for 30 minutes in the dark followed by fixation for 30 minutes with 4% formaldehyde. For protein expression analysis, fixed cells were permeabilized using 0.01% TritonX-100 and then immunostained with anti-GFP primary antibody for 1 hour in the dark after blocking the nonspecific binding with 1% bovine serum albumin blocking buffer. The cells were then rinsed four times with cold PBS followed by incubation for another 1 hour with secondary antibody conjugated to alexa fluor 488 fluorescent dye in order to enable visualization of GFP-positive cells. The cells were imaged at ×10 magnification using Olympus BX63 confocal fluorescence microscope.
Bx63 confocal fluorescence microscope
Manufactured by Olympus
The BX63 confocal fluorescence microscope is a high-performance imaging system designed for advanced fluorescence applications. It features a fully automated confocal optical system and high-resolution image acquisition capabilities.
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2 protocols using bx63 confocal fluorescence microscope
1
Apoptosis Detection in Cells
2
Fluorescence Microscopy of Apoptosis
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In order to determine the morphological changes of the cells after co-treatments, a fluorescence confocal microscopy was employed. Briefly, cells treated with siRBBP6 and pRBBP6 were grown on coverslips mounted into six-well plates and were incubated overnight. This was followed by treatment with 0.25 µM camptothecin or 100 µM GABA, and incubation for further 24 hours. The cells were then washed with cold PBS, and stained with Annexin V-FITC/DAPI followed by fixation with 4% formaldehyde for detection of apoptosis. Furthermore, for protein expression analysis, 0.01% Triton X-100 was added to fixed cells and then immunostained with anti-GFP primary antibody after the cells were washed with 1% BSA blocking buffer. The cells were then washed with cold PBS followed by incubation with GFP-tagged secondary antibody and were imaged at 10× magnification using Olympus BX63 confocal fluorescence microscope.
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