Calcein green am

Polycaprolactone (PCL) (70,000–90,000 g mol⁻¹), Polyvinylpyrrolidone (PVP) (360,000 g mol⁻¹), dichloromethane (DCM) and dimethyl sulfoxide (DMSO) were obtained from Sigma–Aldrich (Switzerland), Polylactic acid (PLLA) (Ingeo™ Biopolymer 2500HP) from Natureworks, N,N- Dimethylformamide (DMF) from VWR Chemicals, Ethanol (without additive, ≥99.8%) and o-xylene from Fluka Analytical, chloroform (CHCl₃, ≥99%) from Fischer Scientific and methanol (CH₃OH, ≥99%) from Fluka. PtTFPP was from Frontier Scientific (USA). Calcein Green AM, cell growth medium, acetone, Na₂SO₃, KH₂PO₄, phosphate-buffered saline (PBS) and all the other reagents were from Sigma-Aldrich (Dun Laoghaire, Ireland). All materials were used without any further purification.

Animals were killed (see above) 1, 2, 3 and 7 days after unilateral nerve transection and a whole mount preparation of the OB was prepared from excised tissue blocks (as described above). The preparation was incubated in bath solution (see “Solutions” section) containing 100 μM Calcein green/AM (Molecular Probes, Thermo Fisher Scientific), 30 μM Alexa 594 dextran (10,000 MW, Molecular Probes, Thermo Fisher Scientific), and 50 μM MK571 (Sigma-Aldrich), an inhibitor of multidrug resistance transporters. Calcein green/AM was initially dissolved in DMSO (Sigma-Aldrich) and Pluronic F-127 (Molecular Probes, Thermo Fisher Scientific). After an incubation time of 2 h, a series of image stacks of the whole ventral OB was obtained using multiphoton microscopy. Relative changes of the OB volume of the transected side to the non-transected side were compared.