Aging Aging

80 Carmody et al.Fig. 4-2. The detection of rod photoreceptor apoptosis in a primary retinal cell cultureusing dual labeling for rhodopsin expression (rod specific protein) and TUNEL.(A) Retinal culture stained with anti-rhodopsin antibody that was subsequently labeledwith a phycoerytherin-conjugated secondary antibody. The encircled rhodopsin-positivepopulation (high FL-2, y-axis) can be readily distinguished from nonrod cells in theretinal culture. (B) TUNEL of rhodopsin-positive cells after 24 h in culture displayinghigh levels of apoptosis. The dashed line represents a negative control of cells stainedwithout the TdT enzyme while the solid line represents TUNEL stained rhodopsinpositivecells.cellular antigen. The cells must therefore be labeled with the specific antibodyfollowing fixation.1. Once the TUNEL assay protocol is completed, incubate cells in appropriatelydiluted (using PBS/1% BSA, w/v) primary antibody for 60 min.2. Wash twice in PBS and once in PBS/BSA (0.1%, w/v).3. Incubate cells in appropriately diluted (using PBS/1% BSA) phycoerythrinconjugatedsecondary antibody for 30 min.