Goat anti mouse igg h l alexa fluor 568

For immunofluorescence staining of nuclear proteins, cells were fixed, permeabilized, and blocked with BSA as described above. The cells were incubated with the following primary antibodies in 1% BSA and 0.03% Triton X-100 in PBS (–) at 4 °C overnight: anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) (5095, Abcam; 1:500), anti-topoisomerase I (85038, Abcam; 1:500), anti-RPA194 monoclonal (48385, Santa Cruz Biotechnology; 1:100), anti-fibrillarin monoclonal (2639, Cell Signaling Technology; 1:500), and anti-NPM 1 monoclonal (32–5200, Invitrogen; 1:500) antibodies. After washing with PBS (–), the cells were incubated with the following secondary antibodies in 1% BSA and 0.03% Triton X-100 in PBS (–) at room temperature for 1 h: Goat Anti-Rabbit IgG H&L-Alexa Fluor^® 568 (175471, Abcam; 1:500) and Goat Anti-Mouse IgG H&L-Alexa Fluor^® 568 (175473, Abcam; 1:500). For nuclear staining, cells were incubated with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) at 1:500 at room temperature for 1 h. For RNA staining, cells were incubated with StrandBrite^TM (AAT Bioquest) at 1:4000 at room temperature for 30 min. For cell viability assays, cells were incubated with 1 μM SYTOX Orange^TM (Thermo Fisher Scientific) and 20 μg/mL bisBenzimide H 33342 trihydrochloride (Hoechst33342; Sigma-Aldrich) in complete medium at 37 °C for 30 min prior to the observation.

EBs were broken down into a single cell suspension using Liberase Blendzyme 2 (0.1 mg/ml, Roche Diagnostics) and fixed in 4% neutral-buffered formalin. After permeabilization with a 0.1% Triton-X solution for 5 min, EBs were double-stained with primary antibodies against CD31 (Abcam, ab28364) and against sarcomeric α-actinin (Abcam). Next, we applied the following secondary antibodies: goat anti-rabbit IgG (H+L) (DyLight 488, ThermoScientific) and polyclonal Alexa Fluor 568 Goat anti-mouse IgG (H+L) (Abcam). FACS analysis was performed with the flow cytometry analyzer (BD FACSAria II).

EBs were fixed in 10% buffered formalin for one hour, and incubated in 0.5% Triton X in PBS at 4 °C overnight. Then, EBs were blocked in PBS with 3% bovine serum albumin for two hours and stained overnight with primary antibodies against CD31 (Abcam, ab28364) and against sarcomeric α-actinin (Abcam). EBs were washed three times with PBS before exposure to secondary antibodies: goat anti-rabbit IgG (H+L) (DyLight 488, ThermoScientific) and polyclonal Alexa Fluor 568 Goat anti-mouse IgG (H+L) (Abcam). After overnight incubation at 4 °C, EBs were washed three times and mounted on glass slides with mounting medium Prolong^R Gold Antifade Reagent (Invitrogen). Mounted EBs were flattened into disks with thickness 20–30 µm on the slides. Finally, EBs were imaged using a laser scanning confocal microscope (Zeiss LSM 700). Specifically, sarcomeric α-actinin was imaged by exciting samples at 555 nm and collecting emission over 560 nm. CD31 was imaged by exciting samples at 488 nm and collecting emission ranging from 488 nm to 550 nm. Positively stained area in individual EBs was quantified using NIH ImageJ software.