Prolong mounting solution

Human thrombin (cell culture grade), fetal bovine serum (FBS), phosphate buffer saline, and bovine serum albumin were purchased from Sigma-Aldrich (St. Louis, MO). Phorbol-12-myristate-13-acetate (PMA), and rottlerin were from EMD/Calbiochem (La Jolla, CA). Anti-PKC (isoform specific), anti-beta-actin and anti-phospho-CPI-17 (Thr38) antibodies were obtained from Santa Cruz Biotech (Santa Cruz, CA); and anti-diphospho-MLC (Thr18/Ser19), anti-total MLC antibodies and anti-phospho-PKD (Ser916) antibodies were from Cell Signaling Technology (Beverly, MA). Anti-mouse and anti-rabbit secondary antibodies conjugated to horse radish peroxidase, enhanced chemiluminescence (ECL), and ECL-Plus were purchased from Amersham Biosciences, Inc /GE Health Sciences (Piscataway, NJ). Texas Red-phalloidin, and Prolong mounting solution were from Molecular Probes (Eugene, OR).

Hippocampal slices with biocytin filled neurons were placed in 4% paraformaldehyde in 0.1 M PB (0.02 M NaH₂PO₄ and 0.08 M Na₂HPO₄) overnight at 4°C. After rinsed in 0.1 M PB (2×10 min), the slices were permeabilized in 2% Triton X-100 (TX-100)/0.1 M PB for 1 hour. They were then incubated in 1% TX-100/0.1 M PB solution with 1 μg/ml avidin-AF488 (Molecular Probes) overnight at 4°C. The slices were then rinsed in 0.1 M PB (2×10 min), and incubated in 300 nM DAPI/0.1 M PB solution for 1 minute. After washed in 0.1 M PB for 10 min, they were mounted on glass slides, and air-dried. The slides were coverslipped with Prolong™ mounting solution (Molecular Probes) and sealed with nail polish. The stained slices were imaged using a Leica SP5X confocal microscope with a 63× oil immersion objective lens. A UV laser was used to excite DAPI signal, and a 488-nm wavelength laser was used for biocytin staining. The CA3 subfield with biocytin filled neurons was imaged through the z-axis at 0.5 μm steps with x/y/z resolution of 0.24/0.24/0.50 μm/pixel.

Cell adhesion onto the alloy surface was analysed using an antibody against vinculin to determine the presence of focal contacts. At the same time, phalloidin was used to visualize actin filaments and their distribution, as reported previously [8 (link)]. The same cell culture protocol described for viability studies was employed, but after 24 h of culture the cells were fixed in 4% PFA in PBS for 30 min at RT, permeabilised with 0.1% Triton X-100 (Sigma) in PBS for 15 min and blocked for 25 min with 1% bovine serum albumin (BSA; Sigma) in PBS at RT. Samples were then incubated with 2 μg/ml mouse anti-vinculin primary monoclonal-antibody (Chemicon, MAB3574) for 60 min at RT and washed with 1% BSA-PBS. Then, samples were incubated with a mixture of 1.4 U/ml Alexa fluor 594-conjugated phalloidin (Invitrogen), 6 μg/ml Alexa fluor 488 goat anti-mouse IgG1 and Hoechst 33258 (both from Sigma) for 60 min at RT. Finally, samples were washed in 1% BSA-PBS, air dried and mounted on specific bottom glass dishes (MatTek) using ProLong mounting solution (Life Technologies). Control analyses were performed in absence of the alloy. Sample evaluation was done with a confocal laser scanning microscope (CLSM, Olympus). One disk was analysed for each surface modification and alloy composition.