Quantifying Renal Treg Cells by Immunofluorescence

For double staining with foxp3 and CD3 (for represent Tregs) [16 (link)], the sections were carried out using paraffin-embedded sections (4 μm) of renal tissues, and incubated with Foxp3 and CD3 primary antibodies individually overnight at 4 °C. The next day, the samples were incubated with rabbit anti-mouse IgG secondary antibody for 1 h at room temperature. The samples were then observed with a fluorescence microscope (IX71, Olympus, Tokyo, Japan) equipped with ISCapture software, and images were taken with a CCD camera (Discovery C15, Olympus, Tokyo, Japan). The numbers of Foxp3⁺ positive (green) and CD3⁺ positive (red) cells counted under fluorescent microscopy.