Carl lsm 510

For histological examination, hematoxylin/eosin staining of paraffin-embedded liver sections was performed as described previously (2 (link)). RAE-1 was detected in immersion fixed frozen sections of mouse liver using Goat Anti-Mouse RAE-1 Pan Specific Antigen Affinity purified Polyclonal Antibody (Clone AF1136) at 5 µg/ml overnight at 4°C. DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) was used and counterstained with hematoxylin (blue).
For fluorescence microscopy, liver cells suspensions from mouse non-treaded or treated with Con A were plated on poly (l-lysine)-coated coverslips (Sigma-Aldrich). Cells were then fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.1% SDS or Triton X-100 for 10 min, followed by blocking with 10% FBS for 20 min. The fixed cells were stained with anti-mouse RAE-1 Pan Specific Alexa Fluor 647-conjugated mAb (Clone #186107), or Rat IgG2A Alexa Fluor 647-conjugared mAbs as isotype control (Clone # 54447), diluted in PBS containing 1 mg/ml BSA. Nuclei were stained with DAPI (Molecular Probes, Invitrogen). Coverslips were mounted with Vectashield (Vector laboratories) and analyzed with a fluorescence microscope (Carl Zeiss LSM-510) and LSM Image Examiner software (Carl Zeiss).

NIH 3T3 cells were treated with recombinant BivCaE protein (50 μg per mL of the medium) or B. terrestris PLA₂ (10 μg per mL of the medium) for 24 h. Subsequently, the NIH 3T3 cells were double-labeled with mouse anti-BivCaE or anti-PLA₂ antibody using an in situ cell death detection kit (Roche Applied Science, Mannheim, Germany). The cell binding assays of the recombinant BivCaE or PLA₂ on the NIH 3T3 cells were carried out by incubation with mouse anti-BivCaE or anti-PLA₂ antibody (diluted 1:200 (v/v)). Subsequently, the NIH 3T3 cells were incubated with tetramethylrhodamine isothiocyanate-conjugated goat anti-mouse Immunoglobulin G (IgG; diluted 1:300 (v/v); Santa Cruz Biotech, Inc., Santa Cruz, CA, USA). Apoptosis assay was performed in NIH 3T3 cells by incubation with the Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) reaction mixture, including terminal deoxynucleotidyl transferase (TdT) and fluorescein-conjugated dUTP. After incubation at 37 °C for 1 h, binding of the recombinant BivCaE or PLA₂ on NIH 3T3 cell surfaces and apoptosis of NIH 3T3 cells were observed using laser-scanning confocal microscopy (Carl Zeiss LSM 510, Zeiss, Jena, Germany).

Cells were fixed and permeabilized with 1% formaldehyde/methanol in PBS for 10 min at room temperature. Next, the cells were washed, and a series of antibodies was used as indicated, followed by staining with FITC- or PE-conjugated goat anti-mouse and anti-rabbit IgG (Calbiochem, San Diego, CA). The samples were then mounted using glycerol, and analyzed by confocal microscope (Carl Zeiss LSM 510; Carl Zeiss, Thornwood, NY) with a 40× C-Apochromat objective. Negative control staining was performed using only secondary antibodies.