Labtek permanox slides

Primary rat motoneurons were isolated as described previously [30 (link)]. Briefly, the ventral horns of the lumbar spinal cord were dissected from Wistar rat embryos (E15). The dissociated motoneurons were plated at a density of 7,000 cells per well on laminin-coated (5 μg/mL; Sigma-Aldrich) 8-well LabTek Permanox slides (NUNC, Denmark). Neurons were stimulated with serially diluted Epobis peptide for 24 h, fixed with 4% formalin, stained with polyclonal rabbit anti-rat growth-associated protein-43 antibodies (1 : 1000; Millipore), and analyzed by computer-assisted fluorescence microscopy as described [29 (link)].

Cerebellar granule neurons were seeded at a density of 62,500 cells/cm² in eight-well poly-d-lysine (0.01 μg/cm²)-coated LabTek Permanox slides (Nunc) and grown for 7 days at 37 °C and 5% CO₂^{82 (link)}. Apoptosis was induced by reducing the potassium levels in the medium from 40 to 5 mM and increasing concentrations of NX-1βe or Neurexide were added. Forty-eight hours later, the cells were fixated, stained and numbers of survived neurons was determined as previously described^{82 (link)}.

To further study the effect of sialyllactose on neuronal survival, primary hippocampal neurons were isolated from Wistar rat embryos (E18; Charles River, Sulzfeld, Germany), plated at a density of 5 × 10⁴ cells/cm² on poly-L-lysine-coated eight-well Lab-Tek^® Permanox slides (NUNC, Roskilde, Denmark) and grown for seven days [51 (link)]. Neurons were then preincubated with serially diluted SAL or lactose for 1 hour, followed by addition of freshly diluted 60 μmol/L H₂O₂ (Sigma-Aldrich). Cells were further cultured for 24 h, fixated with 3.7% (v/v) formaldehyde and 1% (v/v) methanol in PBS, and stained with 5 μg/mL Hoechst 33258 (Invitrogen, Taastrup, Denmark). Images of at least 500 cells/condition were recorded randomly for each group in each independent experiment (n = 4) using computer-assisted fluorescence microscopy [52 (link)]. Data are expressed as means of the live to total cell ratio and were normalized to unstimulated cells.