Goat anti mouse secondary antibody

Cells were grown on a glass coverslip for immunostaining and then provided with hypoxic treatment or the addition of CPO microparticles, as described in the cell culture section. Paraformaldehyde (PFA) (Sigma Aldrich) 4% in PBS for 15 mins was used to fix the cells, followed by permeabilization with 0.05% (v/v) Triton X-100 and blocking with 1% (w/v) BSA (Thermo Scientific) in PBS for 30 mins, all at RT. Next, fixed cells were stained with primary antibodies: Cardiac Troponin T Monoclonal Antibody (13-11), Mouse / IgG1 (Thermo Scientific), and hypoxia-inducible factor 1 (HIF-1α) antibody (H1Alpha67) (Novus Biologicals) for 1 hr. at RT. Following PBS washes, samples were incubated with phalloidin, DAPI, and goat anti-mouse secondary antibodies conjugated (Thermo Scientific) with either Alexa 594 or Alexa 488 (as applicable) for HIF-1α or cTn for 1 hr at RT. After washing with PBS, coverslips were mounted, and fluorescent images were acquired using a Keyence microscope with complementary Image software.

Cells were harvested, fixed, and permeabilized as previously described [16 (link)]. Cells were resuspended in 200 –l of PBS containing 5 –l of primary rabbit anti-SLP-76 or rabbit anti-Vav1 and mouse anti-CD38 antibodies and then stained with Alexa-350 and 430-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies, respectively, from Invitrogen. The immunocomplexes were analyzed using flow cytometry (LSR II, BD Biosciences). The FRET signal was measured as previously described [16 (link)]. The Alexa 350 emission from 325 excitation was measured reflected from a 505 longpass dichroic through a 440/40 bandpass filter. Alexa 430 emission from 488 nm excitation (from an argon ion laser) was collected through a 505 longpass dichroic and 530/30 bandpass filter. Controls with secondary antibody(s) only or secondary(s) plus donor or acceptor primary antibody were included. Cells stained with just SLP-76 or Vav1 or CD38 primary antibody and Alexa 350 or 430 respectively were used for compensation controls for spillover into all fluorescence collection channels. Timing gates on the collected fluorescence defined acceptor emission synchronized to donor excitation. FRET signals were corrected by subtraction of background fluorescence of negative controls with just secondary antibodies and compensation controls.