Ab16434

Embryos were fixed in 4% Paraformaldehyde (PFA) or Dent’s fixative (80% methanol:20% DMSO) and then labeled whole or after vibratome sectioning. For sectioning, embryos were embedded in 5% low-melt agarose (SeaPlaque GTG Cambrex) and sectioned using a 5000 Series Vibratome into 150–200μm sections. Primary antibodies included; mouse anti-desmoplakin I+II (Abcam, ab16434, diluted 1:75), mouse anti α-tubulin (Developmental Studies Hybridoma Bank (DSHB), AA4.3, 1:50), mouse anti-cytokeratin type II (DSHB, 1h5, 1:25), anti-β-catenin (Invitrogen, 71–2700, 1:500), and mouse anti-E-cadherin extracellular domain (DSHB, 5D3, 1:25). Dsp was detected using tyramide amplification (Alexa Fluor 488 Tyramide Superboost, goat anti-mouse, Invitrogen b40941). All other antibodies were detected with anti-mouse or anti-rabbit Alexa Fluor’s used at 1:500 (Invitrogen). Fluorescently labeled substrates included Lectin Peanut Agglutinin (PNA) (Alexa Fluor 488, Invitrogen, L21409, 1:1000) and phalloidin (Rhodamine, Life Technologies, R415, 1:50).

Cells cultured for 8h and 72h on glass coverslips were incubated with BODIPY-C12 for 30min as described above and fixed in 4% paraformaldehyde for 20min at room temperature. Samples were blocked and permeabilized in Block-aid (Life Technologies)/0.1% saponin (Sigma) for 1h before overnight incubation with primary antibody against desmoplakin (1:200, ab16434, Abcam) at 4°C. Following primary antibody incubation, samples were washed with PBS and labeled with secondary antibodies (donkey F(ab’)2 anti-mouse Alexa Fluor 647 (1:500, ab150103), for 1h at room temperature. The samples were washed and counterstained with 1 μg/ml Hoechst 33342 for 10min, mounted in Slowfade Diamond (Molecular Probes), and imaged using a Zeiss 880 LSM Confocal with Airyscan.