Fitc conjugated goat anti mouse igg

PBMCs from 10 randomly selected Sri Lankan donors were evaluated for Class II MHC expression by flow cytometry using anti-DR, -DP, or -DQ antibodies (LB3.1, B7/21, and SPV-L3 clones, respectively). Briefly, thawed PBMCs were counted and the cell suspension adjusted to 1 × 10⁶ cells/ml. Cell suspensions were washed by centrifugation in ice-cold FACS Buffer (PBS, 10% FBS) followed by incubation with unconjugated primary antibodies for 30 min at 4°C. After subsequent wash steps, cells were incubated with FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, Inc. West Grove, PA) for 30 min at 4°C. Additionally, control cells were incubated with isotype antibody only. Data were acquired on a BD FACSCanto™ II analyzer and Median Fluorescence Intensities (MFI) were determined using FlowJo v10 (FlowJo, LLC).

Cumulus-free oocytes were fixed in 4% paraformaldehyde at 37°C for 15 min. Zona pellucida was removed by digestion in 0.5% pronase. Zona-free oocytes were permeabilized in 0.1% Triton X-100 at 37°C for 15 min and blocked for 30 min with 3% BSA. Oocytes were then incubated with mouse Anti-Ceramide antibody (1:50, C8104) or rabbit Active + Pro Caspase-3 antibody (100 fold diluted, ab47131, Abcam) at 37°C for 1 h followed by incubation at 37°C for 1 h with FITC-conjugated goat-anti-mouse IgG (400 fold diluted, 115-095-062, Jackson ImmunoResearch) or Cy3-conjugated goat-anti-rabbit IgG (200 fold diluted, 111-165-144, Jackson ImmunoResearch). Finally, the stained oocytes were mounted on glass slides and observed under a Leica laser scanning confocal microscope (TCS SP2). Argon (488 nm) and helium/neon (543 nm) lasers were used to excite FITC and Cy3, respectively. Fluorescence was detected with 505–540 nm (FITC) and 560–605 nm (Cy3) bandpass emission filters, and the captured signals were recorded as green and red, respectively. Caspase-3 and ceramide expression was quantified by analyzing fluorescence intensity using the ImagePro software.

HASMCs (5×10⁴) were cultured on gelatin-coated coverslips and were cultured in serum-starved medium for 24 h. After a pretreatment with viscolin for 24 h, 30 ng/mL of PDGF-BB and 10 mg/mL of 5-bromo-20-deoxyuridine (BrdU, Sigma-Aldrich, St. Louis, MO, USA) were added for another 24 h. The treated cells were fixed with a 95% ethanol/5% acetic acid solution for 30 min and were treated with 1N hydrogen chloride (HCl) for 10 min after which sodium borate was added to neutralize the HCl. The cells were next incubated with an anti-BrdU antibody (1:100; Sigma-Aldrich) at 4°C overnight followed by a FITC-conjugated goat anti-mouse IgG(1:200; Jackson ImmunoResearch, West Grove, Pennsylvania, USA). The cells were counterstained with 1 μg/mL DAPI (Sigma-Aldrich) and observed under a fluorescence microscope. Six fields were counted under a 20× objective lens to determine the number the number of BrdU-positive nuclei and total number of nuclei (DAPI-positive).