Prior to imaging, RUSH reporter proteins were labeled with JF549 HTL (Janelia Farms) and, for rat neurons, anti-pan-neurofascin antibody (UC Davis/NIH NeuroMab Facility, A12/18; RRID:AB_2877334 ) for 60 min. Coverslips were rinsed twice with warmed PBS and returned to conditioned NBM. Coverslips were incubated with IgG2α Alexa Fluor 647 secondary (Thermo Fisher Scientific, A-21241; RRID:AB_2535810 ) for 15–30 min to label the anti-pan-neurofascin primary antibody. Coverslips were rinsed twice with warmed PBS and imaged in standard extracellular fluid (ECF) imaging solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 5.5 mM glucose, 20 mM HEPES [pH 7.3] in PBS) at 37°C. The reporter proteins were released from the ER-localized streptavidin “hook” with the addition of 40 µM biotin (Sigma-Aldrich, B4639-100MG) to the coverslip. Biotin was diluted in 200 µl of ECF imaging solution and added to 800 µl of media in the imaging chamber for a final concentration of 40 µM. Videos were acquired ~20–30 min after biotin addition at 1 frame per second with a Zeiss 880 Airyscan LSM microscope and 63× objective using Fast Airyscan mode. All images were processed with automatic Airyscan deconvolution settings. Temperature, CO2, and humidity were controlled using an Oko-lab incubation system.
Igg2α alexa fluor 647 secondary
Manufactured by Thermo Fisher Scientific
The IgG2α Alexa Fluor 647 secondary is a fluorescent-labeled antibody used in immunodetection techniques. It is designed to bind to and label IgG2α antibodies, allowing for their visualization and quantification.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using igg2α alexa fluor 647 secondary
1
RUSH Protein Labeling and Live-Cell Imaging
2
Live-cell Imaging of RUSH Reporters
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Prior to imaging, RUSH reporter proteins were labeled with JF549 HTL (Janelia Farms) and, for rat neurons, anti-pan-neurofascin antibody (UC Davis/NIH NeuroMab Facility, A12/18; RRID:AB_2877334) for 60 minutes. Coverslips were rinsed twice with warmed PBS and returned to conditioned NBM growth media. Coverslips were incubated with IgG2α Alexa Fluor 647 secondary (Thermo Fisher Scientific, A-21241; RRID:AB_2535810) for 15-30 minutes to label the anti-pan-neurofascin primary antibody. Coverslips were rinsed twice with warmed PBS and imaged in standard ECF imaging solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 2 mM MgCl 2 , 5.5 mM glucose, 20 mM HEPES (pH 7.3) in PBS) at 37°C. The reporter proteins were released from the ER-localized streptavidin 'hook' with the addition of 40 µM biotin (Sigma-Aldrich, B4639-100MG) to the coverslip. Biotin was diluted in 200 µl of ECF imaging solution and added to 800 µl of media in the imaging chamber for a final concentration of 40 µM. Videos were acquired ~20-30 minutes after biotin addition at 1 frame per second with the Zeiss 880 Airyscan LSM microscope and 63x objective using Fast Airyscan mode. All images were processed with automatic Airyscan deconvolution settings. Temperature, CO 2 , and humidity were controlled using an Oko-lab incubation system.
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