Primary rabbit anti mpo antibody

JEG-3 cells were seeded into 6-well plates on coverslips in growth medium, incubated for 24 h, and subsequently starved overnight. Afterwards, the cells were treated for 4h in starvation medium with 10 μg/ml of MPO (Elastin Products Company) or 5 μg/ml MPO in combination with heparin and 4-ABAH. Heparin and 4-ABAH were added directly prior to MPO treatment. Afterwards, the coverslips were washed with PBS−/−, the cells were fixed with 100% ice-cold methanol for 20 min at −20 °C, and subsequently washed with PBS−/−. Blocking was performed in PBS−/− supplemented with 5% goat serum (Sigma-Aldrich) and 5% BSA (Sigma-Aldrich) for 1 h at RT. A primary rabbit anti-MPO antibody (Cell Signaling, 1:500) in 1:10 blocking solution + 0.1% Triton X-100 was added and incubated overnight at 4 °C. After washing with PBS−/−, the coverslips were incubated with secondary antibody (goat anti-rabbit AlexaFluor488 (Invitrogen, 1:500) for 1 h at RT followed by another washing step. The coverslips were mounted along with DAPI (Vecta Shield Mounting Medium) on glass slides. The cells were observed using an Olympus IX70 fluorescence microscope equipped with a Hamamatsu ORCA-ER digital camera (Hamamatsu Photonics K.K., Japan). CellSense 1.17 Dimension software was used for processing the images.

Paraffin-embedded placenta sections prepared from first trimester elective pregnancy terminations were heated at 60 °C, immersed in xylene, rehydrated in descending alcohol concentrations (100%, 96%, 80%, 70%, 50%), washed in phosphate-buffered saline (PBS) without Ca²⁺ and Mg²⁺ (PBS−/−), and boiled in antigen retrieval buffer (2.94-g/L tri-sodium dihydrate, MW 294 g, pH = 6) for 15 min. Cold antigen retrieval buffer was added and cooled to 4 °C for 1 h. The slides were washed in PBS−/−, incubated for 30 min in 0.3% H₂O₂ (in PBS−/−), and subsequently washed in PBS−/−. The slides were incubated in blocking solution [0.3% TRTX, 5% goat serum, 4% BSA (Sigma-Aldrich) in PBS−/−] at room temperature for 1.5 h and subsequently incubated with primary rabbit anti-MPO antibody (Cell Signaling, clone: E1E7I, 1:500) and primary mouse anti-HLA-G antibody (BD, clone: 4H84, 1:2500) in a humidified box at 4 °C overnight. The slides were washed again and incubated in goat anti-rabbit AlexaFluor488 (Invitrogen, 1:500) and goat anti-mouse AF647 (Invitrogen, 1:500) secondary antibody for 3 h in the dark. The slides were then washed in PBS−/−and background was reduced using Vector True View Kit mix for 3 min. The slides were washed in PBS−/− and the nuclei were stained with Vecta Shield Mounting Medium containing DAPI (Szabo Scandic).