Lsm image browser version 3

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.

Immunostaining of S2 cells and squashed salivary gland polytene chromosomes was performed as described previously [31 (link), 32 (link)]. The primary mouse polyclonal α-dCNDP2 antibodies were used at dilution 1:1000 and detected by goat α-mouse IgG antibodies conjugated to Alexa Fluor 488 (1:500; Invitrogen, A-11001). IF images of S2 cells were made using Zeiss LSM 710 confocal microscope and an oil 100×/1.40 plan apo lens. IF images of polytene chromosomes were acquired with a Zeiss Axio Observer.Z1 fluorescence microscope equipped with an Axiocam 506 mono (D) camera using an oil 63×/1.40 plan apo lens. In both cases, ZEN 2012 software was used for image acquisition.
For detection of eGFP-tagged dCNDP2 fusion proteins, whole salivary glands were fixed in PBS containing 4% formaldehyde (Merck, 104003), washed 3 times for 5 min each with PBS containing 0.5% Triton X-100, stained for 30 min with 0.4 μg/ml DAPI dissolved in PBS and mounted in 50% Glycerol dissolved in PBS. IF images of whole-mounted glands were obtained on a Zeiss LSM 710 confocal microscope using a 20×/0.50 EC Plan-Neofluar lens. Optical sections were combined using the LSM Image Browser version 3.5 software (Zeiss).

All staining procedures were performed according to a previously described protocol [16 (link)]. Tetramethylrhodamine (TRITC)-labeled phalloidin (Sigma-Aldrich, Saint Louis, MO, USA, cat. # P1951) was used at 1:100 dilution to visualize F-actin as described elsewhere [17 (link)]. Primary antibodies were acquired from the Developmental Studies Hybridoma Bank (DSHB): mouse anti-FAS3 (1:10 dilution, clone 7G10); mouse anti-Notch intracellular domain (1:50, C17.9C6); mouse anti-Notch extracellular domain (1:50, C458.2H); mouse anti-Orb antibody (orb4H8, DSHB), 1:30; mouse anti-LaminDmO (ADL67.10, DSHB), 1:30; rabbit anti-p-Histone H3 (Ser 28) antibody (sc-8556, Santa Cruz Biotechnology, Dallas, TX, USA) 1:100; guinea pig anti-Asterless (gift of Tomer Avidor-Reiss, Professor at University of Toledo, Toledo, OH, USA), 1:500. The secondary antibody was a goat anti-mouse IgG antibody conjugated to Alexa Fluor 488 (1:500; Thermo Fisher Scientific, Waltham, MA, USA, cat. # A28175). DAPI was employed (at 1 ng/mL) to stain nuclei. Samples were viewed under an Axioscope 2 plus microscope equipped with ApoTome (Carl Zeiss, Jena, Germany). Optical sections were combined in the LSM Image Browser version 3.5 software (Carl Zeiss) and edited in Adobe Illustrator and Photoshop CS4.