Fitc conjugated affinipure donkey anti rabbit igg

The cervical microarray tissue samples used for the experiment were placed in an oven at 65 °C for 30 min. They were permeabilized in TO bio-clearing agent for 15 min and washed in 100% alcohol, 95% alcohol, and 70% alcohol for 5 min each time. Once the tissue chip was washed, it was placed in a pre-warmed sodium citrate antigen repair solution, heated in a medical microwave oven for 10 min, equilibrated to room temperature, and blocked for 1 h in a phosphate buffer containing 3% bovine serum albumin. Subsequently, it was incubated overnight at 4 °C with primary antibodies against E-cadherin (mouse monoclonal, 1:100, Invitrogen, USA) and CYB5D2 (rabbit polyclonal, 1:50, Santa Cruz Biotechnology, USA). Next, it was washed three times with PBS for 15 min each time. The tissue microarray was incubated with FITC-conjugated AffiniPure donkey anti-rabbit IgG (1:200, Jackson Immuno Research, USA) and Cy3-conjugated AffiniPure goat anti-mouse IgG (1:200, Jackson Immuno Research, USA) for 1 h at 37 °C. Finally, the nucleus was stained with 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI), and fluorescence imaging was performed using a laser confocal microscope (Nikon AIR +, Japan).

The NRK-52E cells (9 × 10⁴ cells/vessel) were grown in eight Chamber Polystyrene Vessel—Culture Slides (Becton Dickinson, Franklin Lakes, NJ, USA). Following the experiment described above, the cells were washed in PBS and fixed in wells with the use of 4% paraformaldehyde in PBS for 12 min at room temperature. Cell membrane permeabilization was performed with 0.2% Triton-X100 in PBS for 10 min in room temperature. Cells were incubated overnight at 4°C with primary antibodies: rabbit polyclonal anti-AT1R N-10 (dilution 1:50; Santa Cruz Biotechnology Inc., Dallas, TX, USA) and goat polyclonal anti-AT2R C-18 (dilution 1:50; Santa Cruz Biotechnology Inc). Next, the slides were incubated for 1 h with Fluorescein (FITC)-conjugated AffiniPure Donkey Anti-Rabbit IgG (for AT1R antibody) and Rhodamine (TRITC)-conjugated AffiniPure Donkey Anti-Goat IgG (for AT2R antibody) secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) diluted 1:50 in the reagent with background-reducing components. Preparations were mounted using Vectashield with DAPI Mounting Medium (Vector Laboratories Inc., Burlingame, CA, USA). The slides were analyzed using BX microscope (Olympus, Tokyo, Japan) coupled with CellF software (Olympus).