Goat anti mouse alexa 488 secondary antibody

In case of transgenes fused with GFP, Paramecia were fixed 10 min with 1% PAF in 1xPHEM, washed in TBST, soaked in TBST containing DAPI for 10 min and mounted on slides using Vectashield (Vector Laboratories). 3xFLAG carrying clones, after fixation, were permeabilized for 4 min with 1% Triton X-100 in 1xPHEM, then washed 2 times in TBST and incubated for 1 h with 1:500 anti-FLAG primary antibody (M2, Merck, F1804), washed 2 times in TBST, incubated 30 min with 1:500 goat anti-mouse Alexa488 secondary antibody (Thermo Fisher. A28175), washed 2 times in TBST and once with TBST containing DAPI. Double stranded RNAs were visualized using 1:100 overnight incubation with mouse anti-dsRNA antibody (J2, SCICONS). Cells were prepared according to the procedure developed for histone modification in (25 (link)).

One day after seeding HDQ-P1 cells at 4,500 cells per well in 96-well plates, the cell culture medium was replaced with fresh medium containing 50 μM G418. A total of 2,658 compounds (Selleckchem 96-well-Z202687-100μl-L1700) were added to each well using a Biorobotics Biogrid II robot equipped with a 0.4-mm diameter 96-pin tool. After 72 h, the medium was removed and cells were rinsed with phosphate-buffered saline (PBS). Cells were then fixed, permeabilized, and stained for nuclei using 3% paraformaldehyde, 0.3% Triton X-100, and 1.5 μg/ ml Hoechst 33323 in PBS for 20 min. Following PBS wash, cells were kept in blocking buffer (3% bovine serum albumin (BSA) in PBS) for 2 h at room temperature. Cells were incubated with mouse anti-p53 (1:1,000, DO-1, Santa Cruz sc-126) in blocking buffer for 1.5 h, rinsed with PBS, and labeled with goat anti-mouse Alexa 488 secondary antibody (1:1,000; Thermo Fisher Scientific) for 1.5 h at room temperature. Cells were then thoroughly washed with PBS, covered with a black membrane, and stored at 4°C until imaging. Imaging was performed using a Cellomics ArrayScan VTI automated fluorescence microscope and 12 fields per well at 20× magnification were captured. The average nuclear p53 immunofluorescence intensity was used for statistical analysis.

5x10⁴ THP1 cells were plated in 96-well plates and treated with 1000 U/mL universal type I IFNα (#11200-2, PBL) or IFNγ (#11500-2, PBL) for 24 h. The next day the cells were treated with the indicated amount of VLPs-Vpx for 16 h. Cells were washed in PBS, and incubated for 15 min at room temperature with the Live/Dead Fixable Blue Dead Cell Stain Kit following instructions provided by the manufacturer (#L34962, ThermoFisher), then fixed in 4% formaldehyde solution (diluted in PBS from a 37% solution, Sigma #252549) for 5 min, and then permeabilized using PBS-Triton X-100 (Sigma, #X100) 0.5% for 15 min. Cells were then stained on ice for 30 min with anti-SAMHD1 antibody (clone I19-18, Millipore #MABF933,) diluted to 0.5 μg/mL in PBS 1% BSA, washed in PBS, and incubated on ice for 30 min with the Goat anti-mouse Alexa 488 secondary antibody (#A-11001, Thermo Fisher), diluted to 4 μg/mL in PBS 1% BSA. Flow cytometry data were acquired using a CANTO-II flow cytometer (BD).

For experiments investigating membrane integrity with the anti-SAG1 antibody, we prepared parasites with the same treatments and conditions noted for replication assays up to primary antibody addition. For staining, we used a monoclonal anti-SAG1 from a 1:1000 dilution of cell supernatant from the DG52 mouse hybridoma, a kind gift from John Boothroyd’s laboratory, combined with a goat anti-mouse-Alexa 488 secondary antibody at 1:750 (Thermo Scientific A-11001). Coverslips were mounted onto slides with Vectashield and imaged on a confocal microscope (ZeissLSM 800 Laser Scanning Microscope).

Worms were fixed in Carnoy’s fixative as described in [78 (link)], were bleached in methanol, and were subsequently stained using the mouse-anti-synapsin primary antibody 3C11 at 1:50 dilution. 3C11 (anti SYNORF1) was deposited to the DSHB by [82 (link)]. A standard immunohistochemistry protocol was used as outlined in [78 (link)], using a goat-anti-mouse-Alexa488 secondary antibody (Invitrogen) at 1:250. Samples were mounted using VetraShield (Vector Laboratories) and imaged on a Nikon AZ100 Multizoom Macroscope or on a Leica SP8.

Asynchronous cell populations were mock-irradiated or irradiated (10 Gy). 6 h or 24 h after irradiation, cells were pulse-labelled with 20 μM BrdU (Sigma) for 20min. After harvesting and fixation in 70% ethanol (4°C), cells were re-suspended in 2 M HCl and incubated for 20 min at RT. Cells were first washed in PBS, then PBS containing 2% fetal bovine serum (FBS; Sigma), before re-suspension in PBS+2% FBS containing mouse anti-BrdU antibody (#347580; BD Biosciences). Cells were incubated in primary antibody for 90 min at RT, washed in PBS+2% FBS and then incubated in goat anti-mouse Alexa 488 secondary antibody (Invitrogen) for 1 h at RT, before re-suspension in PBS containing PI. DNA content and BrdU incorporation were measured using a FACSCalibur Flow Cytometer (BD Biosciences) and BD CellQuestTM Pro Software. Analysis of the data was carried out using the FlowJo software (Tree Star Inc.).