Slowfade gold antifade reagent

Larval tissues were stained with standard immunohistochemical procedures using rabbit anti-phospho-JNK polyclonal antibody (Cell Signaling Technology, Cat #4668, 1:100), chicken anti-β-galactosidase antibody (Abcam, Cat #ab9361, 1:1000), rabbit anti-Cleaved Drosophila Dcp-1 (Asp216) antibody (Cell Signaling Technology, Cat #9578, 1:100), goat anti-rabbit secondary antibody, Alexa Fluor 647 (Thermo Fisher Scientific, Cat #A32733, 1:250) or goat anti-chicken secondary antibody, Alexa Fluor 647 (Thermo Fisher Scientific, Cat #A21449, 1:250). Samples were mounted with DAPI-containing SlowFade Gold Antifade Reagent (Thermo Fisher Scientific, Cat #S36937). Images were taken with a Leica SP8 confocal microscope. The cleaved Dcp-1 positive cell number and the P-JNK-positive area was calculated using ImageJ and Prism 8 (GraphPad).

Dulbecco’s modified minimal essential medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), Trypsin-EDTA solution, 100× penicillin–streptomycin–l-glutamine solution, anti-RFP antibody, Alexa 647-conjugated secondary antibody, SlowFade Gold antifade reagent, and a LIVE/DEAD fixable yellow dead cell stain kit were purchased from Thermo Fisher (Waltham, MA USA). Puromycin solution and G418 solution were obtained from InvivoGen (San Diego, CA, USA). Hygromycin B solution was purchased from Enzo Life Science (Farmingdale, NY, USA). Anti-ORF57, anti-K8, and anti-K8.1 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-LANA and anti-lamin A/C antibodies were purchased from Millipore-Sigma (St. Louis, MO, USA). Anti-nucleolin antibody was purchased from Abcam (Cambridge, UK). Anti-K-Rta antibody was described previously (68 (link)). Monoclonal anti-ORF52 antibody was a generous gift from Fanxiu Zhu (Florida State University). All other chemicals were purchased from Millipore-Sigma (St. Louis, MO, USA) unless otherwise stated.

SaOS and 143B cells (2×10⁴) grown on glass coverslips (pre-coated with 0.01% w/v poly-L-lysine) in 24-well plates were treated with 10 nM eribulin. After 24 hours, cells were fixed with 4% paraformaldehyde, permeabilized using 2% Triton X-100 in PBS, and blocked with 5% horse serum. Cells were immunostained with anti-β-tubulin antibody followed by Alexa Fluor 488-conjugated anti-rabbit IgG (Thermo Fisher Scientific, Rockford, IL) and counterstained with Hoechst 33342 dye (Invitrogen, CA) at a 1:1,000 dilution of the stock solution (10 mg/ml), for 30 minutes at room temperature. The coverslips were mounted in SlowFade Gold Anti-Fade Reagent (Thermo Fisher Scientific, Rockford, IL). Phase-contrast images were obtained using a Leica DM IL microscope equipped with a DFC420 camera and Leica Application Suite Software (Leica Microsystems GmbH, Wetzlar, Germany). Fluorescence images were captured using a Leica TCS SP5 scanning confocal microscope (Leica Microsystems Inc., Buffalo Grove, IL) using the 63× oil objective.

DNA fragmentation in blastocysts that re-expanded by 72 h post-thawing was determined using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction kit assay (In Situ Cell Death Detection Kit with Fluorescein, Millipore Sigma, Burlington, MA, USA). At 72 h post thawing, expanded and hatched blastocysts were collected, washed with PBS-PVP, fixed, and permeabilized as described above. Samples were then separated into three treatments: positive control (DNAse and TUNEL solution), negative control (label solution), and the TUNEL treatment (label solution and enzyme) and subjected to the TUNEL reaction. Briefly, embryos were placed in 25 μl drops of their respective solutions in a four-well plate with water around the wells to allow for a humidified environment. Embryos were placed in a warm incubator for one h, washed in PBS-PVP, then incubated in Hoechst 33342 (1 μg/ml) for 20 min in the dark. Samples were mounted on slides in Slow fade® Gold antifade reagent (Thermo Fisher Scientific) and analyzed by fluorescence microscopy. Excitation/emission was 350/461 for Hoechst 33342 and 520/570 for TUNEL positive cells. ImageJ Software 1.46r (National Institutes of Health) was used to determine the total number of nuclei as well as the number of TUNEL positive (fragmented DNA) nuclei in each embryo (n = 36 controls, n = 38 +FLI, across 5 replicates).

Immunofluorescence staining was performed as described previously. Briefly, cells (5 × 10⁵/well) were seeded on culture slides (Millicell EZ slide, Millipore, ON, Canada) 1 day before staining. At 80% confluence, cells were washed twice with PBS, fixed with fresh 4% formaldehyde for 15 min, penetrated with 0.1% Triton X-100 in 1× PBS for 5 min, incubated with primary antibody for 1 h, washed, and added with fluorescence-conjugated secondary antibody if necessary. Alex Fluor 488 phalloidin (0.15 µM; cat # A12379), mouse anti-ZO-1 antibody (5 µg/mL; cat # 339100), and SlowFade Gold antifade reagent (cat # S36938) were from Thermo Fisher Scientific Inc. Images were acquired by phase-contrast fluorescence microscopy (DMI3000 B, Leica, Wetzlar, Germany) and processed using Q Capture software program.

Cells were fixed with 3.7% (v/v) paraformaldehyde (Electron Microscopy Services, Hatfield, PA) in PBS for 10 minutes and washed 2 × 3 minutes with PBS. Cells were permeabilized with 0.1% Triton X-100 in PBS, followed by a PBS wash. Following blocking in 1% BSA PBS, cells were incubated with AlexaFluor phalloidin 568 (Thermo Fisher Scientific, no. A12380) (5 units/200 μl in 1% BSA PBS per well) for 20 minutes. Cells were washed with PBS three times, wherein the first wash contained DAPI. Cells were mounted in Slow Fade Gold Anti-Fade reagent (Thermo Fisher Scientific) and imaged with a Nikon TE300 inverted fluorescent microscope (Belmont, CA).