Diazabicyclo 2.2.2 octane

Four-day-old embryos generated from incrosses of heterozygous hmx2^SU39 or hmx2;hmx3a^SU44 parents were fixed and processed for phalloidin staining as described in Hartwell et al. (2019 (link)). Stained embryos were stored in DABCO [2% w/v 1,4-Diazabicyclo[2.2.2]octane (D27802; Sigma Aldrich) in 80% glycerol in sterile distilled water].

2,5-Furandicarboxylic acid (FDCA, 98% purity) was purchased from Satachem (Shanghai, China). Di-O-2-(hydroxyethyl) resorcinol (HER) (99%) was obtained from TCI Chemicals (Tokyo, Japan), thionyl chloride (SOCl₂, 99%), 1,4-butanediol (BD, 99%), ethylene glycol (EG, 99%), di-azabicyclo[2.2.2]octane (DABCO, 99%), tin(II) ethylhexanoate (Sn(Oct)₂, 99%) catalysts, and porcine pancreas lipase were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).Triethylamine (Et₃N, 98%) and dimethyl succinate (DMS) were purchased from Panreac (Castellar del Vallés, Spain). Solvents used for reaction, isolation and purification were of high-purity grade and used as received, except dichloromethane (DCM), tetrahydrofurane (THF) and toluene that were dried on 3 Å-molecular sieves. DABCO catalyst was purified by sublimation.

Cultured cells were fixed in 4 % paraformaldehyde followed by 0.25 % triton incubation. Samples were then rehydrated in phosphate-buffered saline, blocked with 1 % bovine serum albumin/10 % normal goat serum, and incubated with primary antibodies for 1 hour at room temperature that included: rat anti-mouse podocalyxin (1 μg/ml; R&D Systems), mouse anti-E-cadherin (0.25 μg/ml; BD Biosciences), rabbit anti-ZO-1 (2.5 μg/ml; Invitrogen-Zymed Laboratories), mouse anti-cytokeratins (clones AE1/AE3, 1:1000; Dako), rabbit anti-ezrin (1 μg/ml; Cell Signaling Technology Inc.), and mouse anti-Muc1 (1:500; gift from Dr John Stingl, Cambridge Research Institute, Cambridge, UK)). Cells were then incubated with fluorescently conjugated species-specific secondary antibodies (Invitrogen-Molecular Probes, Burlington, ON, Canada), and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (Sigma), mounted in glycerol containing the anti-fade agent diazabicyclo[2.2.2]octane (Sigma), and imaged using an Olympus FV1000 confocal microscope (Olympus) followed by processing using FV1000 Fluoview (Olympus) and Adobe Photoshop v12.0 software (Adobe, San Jose, CA, USA).