Cellular Distribution of Fluorescent Colloidal Nanocrystals

Five immunocompetent C57BL6 mice (5 months old, female) were examined to identify the cellular distribution of fluorophore-labeled fCNC. Each animal received a single intravenous dose of fCNC-AF488 and after 24 h, the heart, kidneys, liver, and spleen were harvested, washed in ice cold PBS, and fixed overnight in 4% paraformaldehyde at 4 °C. Fixed tissue was subsequently embedded in paraffin, and sectioned to obtain 0.005 mm samples. Immunofluorescent (IF) staining was performed as previously described^{17 (link),18 (link)} using a Discovery XT processor (Ventana Medical Systems). Briefly, tissue sections were deparaffinized with EZPrep buffer (Ventana Medical Systems), antigen retrieval was performed with CC1 buffer (Ventana Medical Systems) and sections were blocked for 30 min with Background Buster solution (Innovex). The anti-AF488 antibodies (Molecular Probes, cat. no. A-11094, 5 µg/mL) were applied and sections were incubated for 5 h, followed by 60 min. incubation with biotinylated goat anti-rabbit IgG (Vector labs, cat. no. PK6101, 1:200 dilution)^{15 (link),17 (link),18 (link)}. Slides were counterstained with 4′,6-diamidino-2-phenylindole, (DAPI, Sigma Aldrich, 5 μg/mL) for 10 min. and coverslipped with Mowiol. The tissue distribution fCNC was imaged using anti-AF488 IF staining. Images were acquired using Pannoramic Flash (3D Histech) using a 20 × 0.8NA objective.