Anti igg conjugated to fitc

After the SKOV-3 cells were seeded in coverslips and treated with P-MAPA, rhIL-12, or their association for 48 h, they were fixed in methanol for 8 min at room temperature and then washed in sterile ice-cold PBS. To avoid nonspecific bindings, a blocking solution containing 3% (v/v) bovine serum albumin (BSA) was added for 1 h. Then, cells were incubated overnight with rabbit polyclonal anti-TLR2 and anti-TLR4 antibodies diluted 1:200 (Abcam, Cambridge, MA) for 4 h, followed by post incubation with secondary polyclonal anti-IgG conjugated to FITC (1:200 dilution in 1% BSA, Santa Cruz Biotechnology, Inc., CA) for 1 h. After the reactions, 4,6-diamidino-2-phenylindole (DAPI; Sigma, St Louis, MO) was used for nuclei staining. Positive staining was analyzed using a confocal fluorescence microscope Zeiss Axiophot II (Carl Zeiss, Oberköchen, Germany) at different magnification (excitation filter 590 nm, emission filter 650 nm) and for DAPI staining (excitation filter 365 nm, emission filter 485 nm). The relative fluorescence in FITC images was calculated using Image J software.

The OC tissues were isolated, washed in PBS and fixed in 4% paraformaldehyde for 10 min. For tissue permeabilization, proteinase K was used and nonspecific bindings were blocked with 1% BSA for 1 h. Then OCs were incubated overnight with rabbit polyclonal NF-kB p65 antibody diluted 1/100 (ab7970, Abcam, Cambridge, MA), followed by another incubation with secondary rabbit polyclonal anti-IgG conjugated to FITC (1:200 dilution, Santa Cruz Biotechnology, Inc., CA) for 1 h. For nuclei staining, 4,6-diamidino-2-phenylindole (DAPI; Sigma, St Louis, MO) was added (5 min). Positive staining in OC cells was analyzed using a fluorescence microscope (Zeiss Axiophot II, Oberkochen, Germany) at 40× magnification (excitation filter 590 nm, emission filter 650 nm) and for DAPI staining (excitation filter 365 nm, emission filter 485 nm). Relative fluorescence in merged images was calculated using Image J software.