Lung and cell immunostaining were conducted as previously described [50]. For lung tissue, frozen sections were incubated with primary antibodies against CD68 (1:300 dilution) or Ly-6G (1:100 dilution) at 4 °C overnight. For BAL cells, slides with cytospins were incubated with primary antibodies against CD68 (1:300 dilution) after fixation and permeabilized with 0.2% Triton X-100. For RAW 264.7 cells immunostaining, cells were grown in chamber slides with the density of 8.8 × 104 /well and stimulated with silica particles (100 μg/ml). Six hours later, cells were briefly washed with PBS and fixed in 4% paraformaldehyde for 15 min. The slides were then incubated with the primary antibody against F4/80 (1:500 dilution) at 4 °C overnight. In each experiment the slides were then washed with PBS, and reacted with an Alexa Fluor 594-conjugated secondary antibody at room temperature. After nuclear staining with Hoechst 33342, the slides were mounted and scanned by confocal laser scanning microscopy (TiEA1R; Nikon Instech Co., Tokyo, Japan). For localization of FITC signals from silica nanoparticles, imaging software (NIS-Elements AR; Nikon Instech Co.,) was utilized to analyze the fluorescence intensities of silica nanoparticles, F4/80, CD68, and the nucleus. The representative images from at least two independent experiments with biologically duplicated were shown in the result.

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