Lsm5 pascal confocal fluorescence microscope

Immunohistochemistry (IHC) was performed as described previously [48 (link), 50 (link)]. Briefly, the immunized mice (C57BL/6, female) were i.n. challenged with 10 LD₅₀ of PR8, 20 LD₅₀ of CA04, or 10 LD₅₀ of H3N2. Seven days after virus infection, the mice were sacrificed and lung tissues from the mice were obtained, fixed with 10% neutral-buffered formalin, and embedded in paraffin. Formalin-fixed, paraffin-embedded tissue was cut into 4 μm sections and mounted onto glass slides. Tissue slides were blocked with 10% normal goat serum in PBS for 1 h at RT, washed with PBS containing 0.1% Triton X-100 (PBSX), and stained with anti-influenza A NP antibody (Southern Biotech, USA) for 2 h at RT. After washing with PBSX, slides were stained with Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen) for 2 h at RT, followed by staining with 10 μg/ml of 4´,6-diamidino-2-phenylindole (DAPI; Sigma, USA) for 10 min at RT. Then, the stained tissue slides were examined using an LSM5 Pascal confocal fluorescence microscope (Carl Zeiss, Germany).

To examine antigen uptake and processing, 1 μg/mL DQ-OVA (Molecular Probes), which is OVA conjugated to a self-quenched dye that emits green fluorescence upon degradation, was mixed with 400 μg/mL of an adjuvant and incubated for 16 h at 4 °C. DCs were plated onto μ-Slides (Ibidi GmbH) and stimulated with the mixture for 4 h at 37 °C followed by incubating with LysoTracker and DAPI (Molecular Probes). To examine the accumulation of MHC-I in acidic compartments, DCs were incubated with a mixture of OVA-Texas red (1 μg/mL; Molecular Probes) with either alum or PC nanogel for 3 h at 37 °C and then exposed to LysoTracker for the last 30 min. The cells were fixed, permeabilized and stained with an anti-MHC-I (BD) followed by Alexa488-conjugated anti-mouse IgG antibody (Invitrogen). Nuclei were counterstained with DAPI. The stained cells were examined with an LSM5 Pascal confocal fluorescence microscope (Carl Zeiss). The percentages of cells in which MHC-I colocalized with LysoTracker or OVA (Manders’ coefficient) in the insets were calculated using the JACoP plugin of the ImageJ software.