Paraformaldyhyde

After 14 days in culture, samples were fixed in 4% paraformaldyhyde (Electron Microscopy Sciences, Hatfield, PA, USA) containing 100 mM sodium cacodylate trihydrate (Electron Microscopy Sciences) pH 7.4 at 4 °C overnight [19 (link),35 (link),36 (link),38 (link),39 (link)]. Samples were dehydrated with ethanol (KOPTEC, King of Prussia, PA, USA), xylene (VWR, Radnor, PA, USA) infiltrated, and paraffin (Paraplast, Leica Biosystems, Buffalo Grove, IL, USA) embedded. Samples were sectioned to a thickness of 10 µm and stained with Harris Hematoxylin with glacial acetic acid (Poly Scientific, Bay Shore, NY, USA), 0.02% aqueous fast green solution (Electron Microscopy Sciences), and 0.1% Safranin-O solution (Sigma–Aldrich) to visualize condensed nucleic acid material, collagen, and proteoglycans, respectively [19 (link),38 (link)].

Single systems of control and ampullaectomized B. schlosseri colonies were anesthetized using MS-222 (MP Biomedicals, 103106) until oral siphons opened and were unresponsive. Systems were then fixed with 4% paraformaldyhyde (Electron Microscopy Sciences, 15710) with 0.5 M NaCl at 4°C for 12 hours. Following fixation, systems were bleached using 6% H₂0₂ in methanol under light for 1 hour at room temperature to quench auto-fluorescence. Systems were then washed through a series graded methanol and PBS with 0.1% Tween-20 (PBT) and then blocked with 5% heat inactivated horse serum (Jackson ImmunoResearch, 008-000-001) with 2 mg/ml IgG-free BSA (Jackson ImmunoResearch, 001-000-162) in PBT at room temperature for 4 hours. Systems were then incubated with either a Rabbit pan-Cadherin antibody (Cell Signaling, 4068P) (1∶500) or Rabbit pHH3 antibody (Millipore, 06-570) (1∶1000) in blocking buffer and incubated for 48 hours at 4°C. Following primary incubation, system were washed with PBT and incubated with an Alexa Fluor 488 Goat Anti-Rabbit secondary antibody (Life Technologies, A-11008) (1∶200) for 24 hours at 4°C. Systems were then washed with PBT for 6 hours at room temperature to remove unbound secondary antibody and flat mounted in Vectashield mounting medium for fluorescence with DAPI (Vector Labs, H-1200).