Rabbit HMR-1 and HMP-2 antibodies were used at 1 µg/mL. Staining was performed as described previously (Maiden et al., 2013 (link)). Mouse monoclonal GFP antibody was used at 0.2 µg/mL (Molecular Probes).
Microscopy of live embryos was performed using a Nikon Eclipse E600 microscope with a Yokogawa CSU10 spinning disk scanhead. Images were obtained using a Hamamatsu ORCA-ER CCD camera and Micromanager software. 4D movies were processed and analyzed using ImageJ (Abramoff, 2004). Images are Z-projections of 3–4 focal planes spaced 1.0 µm apart. FRAP images were acquired with an Olympus Fluoview FV1000 microscope.
Fluorescence quantitation was performed in ImageJ. Junctions were isolated by thresholding Z projections of 2 focal planes to 99% and selected using the wand tool. Mean fluorescence per pixel for identical seam-dorsal and seam-ventral junctions were compared between embryos. To compare junctional HMP-2::GFP expression to cytoplasmic HMP-2(R271C)::GFP, 20 focal planes of each embryo were sum projected, and mean fluorescence per pixel was calculated for each embryo after subtraction of background signal.
Microscopy of live embryos was performed using a Nikon Eclipse E600 microscope with a Yokogawa CSU10 spinning disk scanhead. Images were obtained using a Hamamatsu ORCA-ER CCD camera and Micromanager software. 4D movies were processed and analyzed using ImageJ (Abramoff, 2004). Images are Z-projections of 3–4 focal planes spaced 1.0 µm apart. FRAP images were acquired with an Olympus Fluoview FV1000 microscope.
Fluorescence quantitation was performed in ImageJ. Junctions were isolated by thresholding Z projections of 2 focal planes to 99% and selected using the wand tool. Mean fluorescence per pixel for identical seam-dorsal and seam-ventral junctions were compared between embryos. To compare junctional HMP-2::GFP expression to cytoplasmic HMP-2(R271C)::GFP, 20 focal planes of each embryo were sum projected, and mean fluorescence per pixel was calculated for each embryo after subtraction of background signal.