Tubulin rat antibody

The primary antibodies used were: Tcf-21 rabbit antibody (Abcam, ab49475; concentration 1:200), β-catenin mouse antibody (BD Biosciences, 610154; concentration 1:400), phospho-paxillin rabbit antibody (Cell Signalling, 2541S; 1:100 concentration), α -tubulin mouse antibody (Sigma-Aldrich, T5168; 1:400 concentration), tubulin rat antibody (Abcam, ab6160; 1:400/1:600 concentration) and diphosphorylated-Myosin Light Chain rabbit antibody (Cell Signalling, 3674S; 1:50 concentration).
The secondary antibodies used were: Alexa Fluor 488 anti-rabbit (ThermoFisher, A21206), Alexa Fluor 555 anti-rabbit (ThermoFisher, A21429), Alexa Fluor 647 anti-rabbit (ThermoFisher, A21245), Alexa Fluor 488 anti-mouse (ThermoFisher, A11029), Alexa Fluor 555 anti-mouse (ThermoFisher, A21424) and Alexa Fluor 647 anti-rat (ThermoFisher, A21247). All secondary antibodies were diluted 1:200.

For STORM imaging, hearts were seeded on glass petri dishes, and F-actin was labeled with phalloidin-647 (ThermoFisher, A22287) at 1:1000 dilution in PBS. Additional immunostaining was performed using the following primary antibodies: Paxillin mouse antibody (BD Biosciences, 610051) and tubulin rat antibody (Abcam, ab6160). Additional secondary antibodies were: Alexa Fluor 488 anti-rat (ThermoFisher, A11006) and Alexa Fluor 555 anti-mouse (ThermoFisher, A21424). All antibodies were diluted 1:1000.
Images were acquired using a Nikon N-STORM 4.0 system configured for total internal reflection fluorescence (TIRF) imaging. Excitation inclination was tuned to adjust focus and to maximize the signal-to-noise ratio. For STORM imaging Alexa647 was excited illuminating the sample with the 647 nm (∼160 mW) laser line built into the microscope. Fluorescence was collected by means of a Nikon ×100, 1.4 NA oil immersion objective and passed through a quad-band-pass dichroic filter (97335 Nikon). Images were recorded on a 256×256 pixel region (pixel size 160 nm) of a sCMOS camera (Hamamatsu). Samples were kept in Gloxy buffer for STORM imaging as previously described 57 . Single-molecule localization sequences were analyzed with the STORM plug-in of NIS element Nikon software.