Slowfade diamond antifade

To detect cells undergoing apoptosis in retinal capillaries, terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL) assay was performed using a kit (ApopTag Fluorescein In Situ Apoptosis Detection; Millipore Sigma) as described previously [39 (link)]. Briefly, RTDs were fixed in paraformaldehyde, permeabilized in a pre-cooled mixture of a 2:1 ratio of ethanol/acetic acid, washed in PBS, exposed to equilibration buffer, and incubated for 1 h with deoxyribonucleotidyl transferase (TdT) enzyme in a moist chamber at 37 °C. Following incubation, RTDs were exposed to anti-digoxigenin peroxidase, washed in PBS, counterstained with DAPI, and mounted using anti-fade reagent (SlowFade Diamond Antifade, Cat#S36963; Invitrogen, Carlsbad, CA, USA). At least five images representing random fields of the RTD slide were captured using a digital microscope (Nikon Eclipse; TE2000-S) and TUNEL-positive cells per total number of cells per field were analyzed.

For immunostaining, cells were generally fixed in 2% formaldehyde and permeabilized as described earlier [13 (link)]. For immunostaining of Ku80 we followed the protocol of Britton, et al. [44 (link)] with some changes: Pre-extraction with CSK+R buffer was performed 3x instead of 2 × 3 min, fixation occurred for 15 min with 4% instead of 2% formaldehyde, and permeabilization was done for 10 min instead of 5 min with PBS, 0.2% Triton X-100. Primary antibodies for immunofluorescence staining were diluted in 1x PBS, 0.4% BSA: α53BP1 (Ab-1) (rabbit, Calbiochem, PC712, 1:500), αCENP-F (rabbit, Novus, NB500-101, 1:750), αCtIP (rabbit, Bethyl Laboratories, A300-488A, 1:100), αGeminin (mouse, clone 1A8, Novus Biologicals, H00051053-M01, 1:100), αγH2AX (Ser 139) (mouse, clone JBW301, Millipore, 05-636, 1:500), αKu80 (mouse, clone 111, Thermo Scientific, MA5-12933, 1:100), αRPA/P34 (mouse, clone 9H8, Sigma, R1280, 1:3000). To visualize the primary antibodies, secondary Alexa 488-, Alexa 568-, or Alexa 647-conjugated αmouse or αrabbit antibodies from donkey or goat were used (Invitrogen, Life Technologies, Sigma-Aldrich). DNA was counterstained with DAPI (AppliChem; 1 µg/mL). In the end, the samples were mounted in Slow Fade Diamond Antifade (Invitrogen, Waltham, MA, USA) and analyzed at a Leica TCS or Nikon spinning disk microscope.

Fluorescent orange beads with a diameter of 200 nm (FluoSpheres, Invitrogen) suspended in water were dropped on a coverslip and dried. The inverted coverslip was placed on a glass slide with a small amount of water and sealed with nail polish. The prepared slide was used for the PSF measurement and the calibration process. For the 3D imaging demonstration, fluorescent beads sparsely distributed in agarose gel were used. For this purpose, the same 200-nm beads were embedded in agarose gel (1% agarose L, Nippon Gene), which was mounted on a glass slide using a slide seal and covered by a coverslip.
COS-7 cells (DS Pharma Biomedical) were cultured on a coverslip with Dulbecco’s modified Eagle’s medium (Fujifilm Wako Pure Chemical) supplemented with 10% foetal bovine serum in a CO₂ incubator at 37 °C. The cells were then fixed with 4% paraformaldehyde and stained with Alexa Fluor 532 phalloidin (A22282, Invitrogen). The stained cells were mounted on a glass slide with an antifade reagent (SlowFade Diamond Antifade, Invitrogen).

RAW264.7 cells were seeded at 2 × 10⁵ cells per well in a 24-well plate with 10-mm coverslips and allowed to attach overnight at 37°C and 5% CO₂. Infection with Syto9 fluorescent-labeled E. faecalis V583 was performed with MOI of 10 for 1 hour. The coverslips seeded with cells were then fixed with 4% PFA at 4°C for 15 min, permeabilized with 0.1% Triton X-100 for 15 min at room temperature, and washed thrice in PBS. Cells were then blocked with PBS supplemented with 0.1% saponin and 2% BSA. For actin labeling, the phalloidin–Alexa Fluor 555 conjugate (Thermo Fisher Scientific, USA) was diluted 1:40 in PBS and incubated for 1 hour. Coverslips were then washed three times in PBS with 0.1% saponin. They were then subjected to a final wash with PBS, thrice. Last, the coverslips were mounted with SlowFade Diamond Antifade (Thermo Fisher Scientific) and sealed. Confocal images were then acquired on a 63×/numerical aperture (NA) 1.4, Plan Apochromat oil objective fitted onto Elyra PS.1 with LSM 780 confocal unit (Carl Zeiss) using the Zeiss Zen Black 2012 FP2 software suite. Laser power and gain were kept constant between experiments. Z-stacked images were processed using Zen 2.1 (Carl Zeiss). Acquired images were visually analyzed using ImageJ (52 (link)).