Mouse monoclonal anti tnnt2 antibody

Immunocytochemistry was performed as previously described^{4 (link)}. Briefly, cells were fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature, permeabilized with 0.1% Triton X-100 and blocked with 10% goat serum. Treated cells were then incubated with mouse monoclonal anti-Tnnt2 antibody (1:500; Thermo Scientific, MA5–12960), rabbit anti-GFP antibody (1:500; Thermo Scientific, A-11122) or mouse anti-α-actinin (1:500; Sigma, A7811) in 1% goat serum at 4 °C overnight. After three washes with PBS, cells were incubated with Alexa Fluor secondary antibodies (1:500; Invitrogen) for 1 h at room temperature. Image acquisition was performed using a BZ-X710 or BZ-X800 (Keyence). Cells were manually quantified in ten randomly selected low-power fields of view from each well in three independent experiments. Results were then averaged to yield an individual replicate.

Immunocytochemistry was performed as previously described (Zhou et al., 2015 (link)). Briefly, cells were fixed in 4% PFAfor 15 min at room temperature and blocked with 5% goat serum. Fixed cells were then incubated on a rotator with mouse monoclonal anti-Tnnt2 antibody (1:500, Thermo Scientific, MA5–12960), rabbit anti-GFP antibody (1:500, Thermo Scientific, A-11122), and rabbit anti-mCherry antibody (1:500, Abcam, ab167453) in 5% goat serum for 1 h at room temperature or 4 °C overnight. After three washes with PBS, cells were incubated with appropriate Alexa fluorogenic secondary antibodies (1:500, Invitrogen) at room temperature for 1 hr. Image acquisition and analysis was done on a BZ-X710 (Keyence). For quantification, cells were manually quantified and averaged to yield an individual replicate in four randomly selected low-power fields of view from each well in three independent experiments.

Flow cytometry was performed as previously described^{8 (link)}. Briefly, reprogrammed iCLMs were trypsinized using TrypLE Express (Gibco) and resuspended into a single-cell suspension. Then, cells were fixed and permeabilized using BD Cytofix/Cytoperm (BD Biosciences). Cells were stained with rabbit anti-GFP antibody (1:200; Thermo Scientific, A-11122) and mouse monoclonal anti-Tnnt2 antibody (1:200; Thermo Scientific, MA5–12960) and secondarily stained using donkey anti-mouse Alexa Fluor 647 (1:200; Invitrogen, A-31571) and goat anti-rabbit Alexa Fluor 488 (1:200; Invitrogen, A-11008). Flow cytometry was performed using a FACSCalibur instrument (BD Biosciences) and analysis was performed using FlowJo software.