Inverted sp5 confocal microscope

For confocal microscopy experiments, approximately 1 X 10⁶ cells were allowed to adhere to fibronectin-coated glass coverslips for 2–3 hours. Thereafter, cells were washed with wash buffer containing phosphate buffered saline (PBS, Invitrogen/Life Technologies), 0.5% human serum albumin, 4 mg/mL IVIG and 0.1% Tween-20 and incubated with pre-formed hPF4 ± heparin in M199 media, for varying times at 37°C. To inhibit actin polymerization, cells were incubated with 5 μM cytochalasin D for 1 hour at 37°C along with labeled complexes and then incubated for 24 hours. To inhibit macropinocytosis, cells were pre-incubated with 0.1 mM amiloride in media for 15 minutes prior to addition of labeled complexes. After incubation with antigen, cells were washed and fixed with 4% paraformaldehyde and counterstained with one or more of the following labeled-antibodies: CD14 (monocyte marker), CD1a (dendritic cell marker), KKO (anti-hPF4/heparin monoclonal antibody, mAb), and DAPI (nuclear stain). Coverslips were mounted on slides with Fluoromount-G mounting medium, and examined and photographed with a Leica SP5 inverted confocal microscope using a 100X oil immersion lens (Leica Microsystems, version-LAS AF 2.6; Wetzlar, Germany), with digital zoom (2-10X) and accompanying Leica LAS AF 2.6 software (Buffalo Grove, IL).

An SP5 inverted confocal microscope (Leica Microsystems) was used to image fluorescent transgenic embryos, double fluorescence in situ hybridization and TUNEL assays. Fluorescein and GFP were excited by a 488-nm laser, DsRed and Cy3 by a 543-nm laser, Strepavidin-647 by a 633-nm laser and 4,6-diamidino-2-phenylindole (DAPI) by a 405-nm laser. Sequential images were overlaid using ImageJ (NIH) or Imaris (Bitplane). A Zeiss AxioZoom.V16 microscope was used to image the immunohistochemistry samples using the AxioVision software 4.8. Visible-light imaging for sections or WISH was performed on a BX-51 Olympus or Leica MZ16 microscope (Leica FireCam Software 3.4.1).