Immunocytochemistry assays were performed in 8-well slide chambers. Briefly, cells were fixed in 4% paraformaldehyde (PFA) for 20 minutes, permeabilized with Triton X-100 0.1% in PBS, and non-specific epitopes were blocked in 10% bovine serum albumin (BSA) in 1 × PBS for 1 hour at room temperature. Immunostaining was accomplished by incubating primary antibodies overnight at 4 °C. Samples were incubated for 2 hours at room temperature with Alexa Fluor 555 conjugated secondary antibodies diluted at 1 in 250 (MeridianLife Science Inc., Memphis, TN), and nuclei were stained with Hoechst (2 μM Hoechst33258). Pictures were taken with a Zeiss LSM 510 confocal microscope and a Zeiss Axioplan-2 deconvolution microscope.
To assess cell death, primary human RPE cells and ARPE-19 cells were fixed with methanol for 15 minutes, washed with 1 × PBS, then loaded with 2 μM Hoechst dissolved in a Locke’s solution (Promega) and incubated for another 15 minutes before imaging. Cells were then viewed by using a Zeiss LSM 510 confocal microscope under UV fluorescence. Images were recorded, and cell apoptosis was assessed by using an automated unbiased method70 (link).
To assess cell death, primary human RPE cells and ARPE-19 cells were fixed with methanol for 15 minutes, washed with 1 × PBS, then loaded with 2 μM Hoechst dissolved in a Locke’s solution (Promega) and incubated for another 15 minutes before imaging. Cells were then viewed by using a Zeiss LSM 510 confocal microscope under UV fluorescence. Images were recorded, and cell apoptosis was assessed by using an automated unbiased method70 (link).
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