Example 6
Immunocytochemistry and Cell Apoptosis Assessment:
Immunocytochemistry assays were performed in 8-well slide chambers. Briefly, cells were fixed in 4% paraformaldehyde (FA) for 20 mins, permeabilized with 0.1% Triton X-100 in PBS. Non-specific epitopes were blocked in 10% Bovine serum albumin (BSA) in 1×PBS for 1 h at room temperature. Immunostaining was by incubating primary antibody overnight at 4° C. Samples were stained for 2 h at room temperature with Alexa Fluor 555-conjugated secondary antibodies diluted 1 in 250 (MeridianLife Science Inc., Memphis, Tenn., USA), and nuclei were stained with Hoechst (2 μM Hoechst 33258). Pictures were taken with a Zeiss LSM 510 confocal microscope and a Zeiss Axioplan-2 deconvolution microscope.
To assess cell death, hRPE and ARPE-19 cells were fixed with methanol for 15 mins, washed with 1×PBS then loaded with 2 μM Hoechst dissolved in a Locke's solution (Promega) and incubated for another 15 mins 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 method.