Dmra2 fluorescence microscope

GFP-Pc iRBC-infected B6 mice were sacrificed and PBS-perfused. Spleens were removed and frozen in Tissue-Tek OCT (Sakura Fineteck, Japan). Sections 8 µm thick were cut with a CM3050S Cryostat (Leica, USA) and fixed with 1% paraformaldehyde (Alfa Aesar, USA) for 30 min at RT. Sections were incubated with anti-CD16/CD32 mAb (Fc block; BD Biosciences) for 30 min followed by incubation in a humidified dark chamber with fluorescent mAbs against CD11c, CD19, CD3, CD4 (BD Biosciences) and MOMA-1 (Abcam) for 2 h at RT. Sections were then stained for 5 min with 0.5 μg/ml DAPI (4',6-diamidino-2-phenylindole; Sigma-Aldrich), washed with PBS and mounted with Fluoromount-G (Southern Biotechnologies, USA). Images were acquired with a DMRA2 fluorescence microscope (Leica) and MetaMorph software (Molecular Devices Inc., USA). Image analysis was performed with Photoshop CS4 (Adobe Inc., USA). Percentages of CD11c-GFP/CD11c-CD4 pixel colocalization and of GFP pixel distribution in the spleen were calculated using FIJI for Windows 64-bit (Colocalization threshold and Mixture Modeling Thresholding plugins, respectively; General Public License, NIH, USA).

Statistical analysis was performed with SPSS 13.0 statistical software (SPSS Inc., Chicago, IL, USA). Results were expressed as the mean ± SE of three or more observations (as indicated in each experiment). Differences amongst the three groups were assessed using one-way ANOVA and differences between the two groups were assessed using the Student-Newman-Keuls (SNK). A p value equal to or less than 0.05 was considered statistically significant.Fig. 2

Binding of Peptide 1 to the cell surface (×280). Immunofluorescence was used to stain Peptide 1 binding to HO8910 cells. Cells were visualized using a Leica DMRA2 fluorescence microscope. a and b HO8910 cells. c and d OSE cells

Vero cells were grown directly on slides in a Nunc Lab-Tek II Chamber Slide System to a confluence of 10⁴ cells/well. The cells were transfected with liposomes at an 8 nM molar ratio with 6 µg and 2 µg of RNA replicons expressing GFP, and a control well was transfected only with liposomes. Transfection was performed in the same manner as in Section 2.5 in this section, only in a smaller volume (0.5 mL instead of 2 mL per well). After 48 h of transfection, the sections were stained with 0.03 µg/mL DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich, St. Louis, MO, USA), washed with DMEM medium, and the slide was separated for microscopic examination. Figures were acquired using a DMRA2 fluorescence microscope (Leica, Wetzlar, Germany) and MetaMorph software (Molecular Devices Inc., San José, CA, USA), and edited using Figure J (Version 1.51n).