Alexa fluor 647 goat anti mouse igg h l antibody

Primary antibody sources were as follows: mouse monoclonal mCherry antibody (diluted 1:1,000 for western blotting, 1:50 for EM, ab125096), rabbit monoclonal CD9 antibody (diluted 1:50 for EM, ab92726) and NF-κB antibodies (diluted 1:1,000 for immunocytochemistry, ab16502) were obtained from Abcam (Cambridge, MA, USA); CD63 (diluted 1:1,000 for western blotting, sc-15363) and GAPDH (diluted 1:4,000 for western blotting, sc-25778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); cytochrome c antibody (diluted 1:1,000 for immunocytochemistry, 556432) was from BD biosciences (San Jose, CA, USA); and the GFP antibody (diluted 1:1,000 for western blotting, 2555) was from Cell Signaling Technology (Beverly, MA, USA). Secondary antibody sources were as follows: anti-rabbit (sc-2004) and mouse secondary antibody (sc-2005) were obtained from Santa Cruz Biotechnology, and Alexa Fluor 647 goat anti-mouse IgG (H+L) antibody (diluted 1:1,000 for ICC, A-21235) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) antibody (diluted 1:1,000 for ICC, A-11008) were purchased from Thermo Fisher Scientific (Wilmington, DE, USA).

Immunofluorescence was performed as previously described (Cronan et al., 2016 (link)). Briefly, zebrafish larvae were fixed in Dent’s fixative overnight at 4°C, rehydrated in PBS containing 0.5% tween 20, and then blocked for 1 h in PBDTxGs (PBS containing 1% BSA, 1% DMSO, 0.1% Triton X-100, 2% goat serum). Mouse anti-E-cadherin antibody, clone 36 (BD, 610181) was added at a 1/500 dilution followed by incubation overnight at 4°C. Larvae were washed in PBDTxGs and then Alexa Fluor 647 Goat Anti-Mouse IgG (H+L) antibody (ThermoFisher, A-21236) added at a 1/500 dilution followed by incubation overnight. Larvae were washed 5 times in PBDTxGs before analysis.