4 6 diamdino 2 phenylindole dihydrochloride dapi

Free-floating fluoroimmunohistochemistry of the lung was performed with 60 μm-thick sections floating in solution in a 48-well plate. The sections were fixed with 4% paraformaldehyde phosphate buffer, permeabilized with 1% Triton, blocked with 10% goat serum (DAKO, Carpinteria, CA), and then stained with a rabbit anti- MMP-12 antibody (Abcam plc, Cambridge, UK) and FITC-conjugated rat anti-F4/80 antibody (eBioscience). After washing three times with 0.2% Triton, the sections were stained with Alexa 568 goat anti-rabbit immunoglobulin (Ig)G and Alexa 488 goat anti-FITC-IgG (Invitrogen Corporation, Carlsbad, CA). Nuclear DNA was stained with 4′,6-diamdino-2-phenylindole dihydrochloride (DAPI) (Invitrogen Corporation). Sections were observed using a PASCAL confocal laser-scanning microscope (LSM: Carl Zeiss, Jena, Germany) at 400× magnification. LSM image browser version 3.5 (Carl Zeiss) was used for image acquisition. The number of positive cells per square millimeter was calculated.

Frozen sections of lacrimal gland tissues were fixed in 4% paraformaldehyde in phosphate buffer, blocked with Blocking One Histo (Nacalai Tesque, Kyoto, Japan), and then stained with Alexa 546-conjugated rat anti-mouse CD4, CD8, CD19, or F4/80 monoclonal antibodies (eBiosciences, San Diego, CA, USA) overnight. After washing three times with PBS, nuclear DNA was stained with 4′,6-diamdino-2-phenylindole dihydrochloride (DAPI, Invitrogen, Carlsbad, CA, USA). Sections were observed with a PASCAL confocal laser-scanning microscope (LSM: Carl Zeiss, Jena, Germany) at 400× magnification. The LSM image browser version 3.5 (Carl Zeiss, Oberkochen, Germany) was used for image acquisition.

Frozen sections (6 μm) of salivary gland tissues were fixed with cold acetone, blocked with 10% goat serum (DAKO), and then stained with a rabbit monoclonal antibody against CCL22/MDC (abcam), FITC-conjugated anti-mouse F4/80 (BioRad), rat monoclonal antibody against anti-EpCAM (eBioscience, G8.8), anti-CD3 (eBioscience, 1.45-2C11), anti-CD19 (eBioscience, eBio1D3), and biotinylated anti-CD11c (Biolegend, N418) antibodies. After washing with PBS, Alexa Fluor 568-conjugated anti-rabbit IgG (Invitrogen) and Alexa Fluor 488-conjugated anti-fluorescein Green goat IgG fraction (Invitrogen), or Alexa Fluor 488-conjugated anti-rat IgG (Invitrogen), or Alexa Fluor 488-conjugated streptavidin (Invitrogen) were used as secondary antibodies. Nuclear DNA was stained with 4′,6-Diamdino-2-phenylindole dihydrochloride (DAPI) (Invitrogen). In addition, the paraffin-embedded sections from SS patients and controls were stained with anti-CD68 (DAKO, Klon EBM11), anti-Keratin (DAKO, AE1/AE3), anti-CD3 (DAKO, F7.2.38), anti-CD19 (DAKO, LE-CD19), anti-S100 (abcam, 4C4.9), and anti-CCL22 Abs (abcam). The sections were examined using a PASCAL confocal laser-scanning microscope (LSM: Carl Zeiss) at 400 × magnification. LSM image browser version 3.5 (Carl Zeiss) was used for image acquisition.