A31571

Cells were plated in 384-well CellCarrier plates (PerkinElmer, Waltham, MA) at a concentration of 60 cells/μl (3000 cells/well) and incubated for 24 h before IF staining. Cells were fixed in 4% PFA in PBS for 15 min, washed for 5 min three times with PBS, and permeabilized in 0.5% Triton X-100/PBS for 15 min at room temperature. Cells were then washed for 5 min three times with PBS, blocked for 20 min with 3% bovine serum albumin/PBS, and incubated with the indicated primary rabbit or mouse antibodies at the indicated concentrations (Supplemental Table S1) and anti–cyclin-A2 antibody (1:125; clone 6E6; ab16726; Abcam) for 30 min at room temperature and washed for 5 min three times with PBS before a 30-min incubation with appropriate anti-rabbit or anti-mouse secondary antibodies labeled with Alexa 488 (1:200; A11034) or Alexa 647 (1:200; A31571; Life Technologies). After three 5-min washes with PBS, DNA was counterstained with 10 μg/ml DAPI for 10 min at room temperature. Cells were left in 100 μl/well PBS and either immediately imaged or stored at 4°C in sealed plates until imaging. In all experiments, one histone or histone modification was detected per well using manual staining.

The rat retinas were digested in a solution containing papain and dissociated as described above. In all steps, including suspension, washing and incubation, an AO⁺ culture medium was used. Mouse IgG1κ antibody (553447; BD Biosciences, San Jose, CA, USA) was used as an isotype control. The cells were re‐suspended and reacted with mouse anti‐Thy‐1.1 antibody (1 : 20; MAB1406; Millipore Corporation) at 25°C for 30 min. After washing twice, the samples were incubated with an Alexa Fluor 647 donkey anti‐mouse immunoglobulin (IgG) secondary antibody (1 : 200; A31571; Life Technologies Inc.) at room temperature for 30 min and again washed twice. For double staining, the cells were incubated with mouse anti‐CD31 antibody (1 : 10, 550300; BD Pharmingen, San Jose, CA, USA) pre‐labeled with Zenon Alexa Fluor 350 (Life Technologies Inc.) at 25°C for 30 min. After washing twice, the cells were re‐suspended in medium containing 0.5% 7‐aminoactinomycin D (Life Technologies Inc.) to exclude dead cells, and were immediately sorted using fluorescence‐activated cell sorting with the Aria II device (BD Biosciences). The cells were sorted, placed into 350 μL of QIAzol Lysis Reagent (Qiagen, Hilden, Germany) and analyzed with qRT‐PCR. The number of sorted cells in each fraction was matched, and the same number of pre‐sorted cells was used as a reference for klotho expression in the naïve rats.

HEK-293 cells were grown in poly-d-lysine–coated (Sigma-Aldrich) coverslips. After treatment, the cells were rinsed with PBS twice, fixed for 10 min in 4% paraformaldehyde, and permeabilized for 10 min with 0.1% Triton X-100 and 100-mM glycine in PBS. The cells were blocked for 60 min with PBT (PBS + 1.5% BSA and 0.1% Tween 20). Primary antibodies used were rabbit anti–TDP-43 (1:500; 10782-2-AP; Proteintech Group, Inc.), mouse anti–TIA-1 related protein (TIAR; 1:500; BD), and mouse anti–α-smooth muscle actin (1:100; A2547; Sigma-Aldrich). Secondary antibodies used were goat anti–mouse IgG, DyLight 488, goat anti–rabbit IgG, DyLight 594 (both 1:750; Thermo Fisher Scientific), and donkey anti–mouse IgG Alexa Fluor 647 (1:750; A31571; Life Technologies). Primary antibodies were diluted in PBT and incubated with the coverslip for 60 min. After washing the cells twice with PBS, they were incubated with the secondary antibody for 60 min in the dark. Cells were washed three times with PBS and mounted in mowiol containing DAPI.