The media was replaced every two days with re addition of apoE. At 8 DIV, the cultures are fixed, immunostained for tubulin selleck products III, and neurite out growth measured as described above. For studies with LRP inhibitors, lactoferrin was obtained from Sigma Chemical, and purified recep tor associated protein was generously provided by Dr. Dudley Strickland. The OE cultures at 2 DIV were pre incubated for 1 h with medium alone or with either RAP or lactoferrin. Following incubation, apoE isoforms were added to the medium. The media was replaced every two days with re addition of the test reagents. At 8 DIV, the cultures are fixed, im munostained Inhibitors,Modulators,Libraries for tubulin III, and neurite outgrowth measured as described above. Immunocytochemistry Immunostaining of neuronal marker, tubulin III, was performed as previously described.

Briefly, cover slips of cultures at 8 DIV were rinsed with warm phos phate buffered saline and fixed with 4% parafor maldehyde in PBS for 15 min at room temperature. Cells were permeabilized with 0. 5% Triton in PBS Inhibitors,Modulators,Libraries for 10 minutes. Cells were rinsed with PBS and blocked with 5% donkey serum and 0. 05% Triton in PBS for 60 minutes. Cells were incubated with mouse anti tubulin III at 1 200 dilution in blocking solution for 2h at room temperature. Following incuba tion, cells were rinsed three times with PBS and incubated with TRITC conjugated donkey anti mouse in blocking solution at 1 200 Inhibitors,Modulators,Libraries for 1h. Cells were rinsed with PBS and were mounted with mounting medium. Immunoreactive cells were counted and photographed on an Olympus BX50 microscope with appropriate excitation filters.

Immunocytochemistry of olfactory Inhibitors,Modulators,Libraries sensory neurons using markers for mature and immature was performed as described above for tubulin III. At 8 DIV cells were fixed, permeabilized, and blocked with 1% BSA for 10 minutes. Cells were incubated overnight at 4 C with goat anti OMP at 1 500 dilu tion in 4% donkey serum or with rabbit anti GAP43 at 1 200 dilution in 4% rabbit serum. Following incubation, cells were rinsed with PBS and incubated for 1h at room temperature with Cy3 conjugated donkey anti goat at 1 500 dilution in 4% donkey serum Inhibitors,Modulators,Libraries or with FITC conjugated donkey anti rabbit at 1 1000 dilution in 4% rabbit serum. Cells were washed, mounted on glass slides, and photographed as described above for tubulin III. All controls with no primary were negative.

Statistical analysis All experiments were repeated at least four times using different preparations of OE cultures and reagents. The data in individual experiments were presented as mean standard error, and statistical analysis was performed using Sigmaplot software. Results and discussion Characterization of olfactory epithelium selleckchem Nilotinib cultures We used a modified protocol to culture olfactory epithe lium cells derived from post natal mice. At 4 days in vitro the neuronal precursors and sensory neurons migrate out of the explant and establish as two large halos.