Anti cd80

Kidney samples were obtained from excess tissue corresponding to kidney nephrectomy specimens donated to the biobank of the IIS-Fundacion Jimenez Diaz Biobank after diagnostic evaluation was performed. The local Ethics Committee approved the study protocol and informed consent was obtained. Control human kidney specimens were taken from normal portions of renal tissue from patients who underwent surgery because of localized renal tumors. One renal biopsy from a male Fabry patient was studied, a 69 year old with serum creatinine 4.2 mg/dl, proteinuria 0.7 g/24 h. Immunohistochemistry was carried out in paraffin-embedded tissue section 5 μm thick. The primary antibody was mouse monoclonal anti-CD80 (1:100, Abcam). Sections were counterstained with Carazzi’s hematoxylin. Negative controls included incubation with a non-specific immunoglobulin of the same isotype as the primary antibody.

Paraffin-fixed tissue samples were cut into sections of a 5-μm thickness and stained with hematoxylin and eosin (HE) for histological examination. Immunofluorescence staining was further performed to evaluate the histomorphological expression features. Sections were incubated with primary antioccludin (1:50; Santa Cruz), anti-ZO-1 (1:50; Santa Cruz), anti-CD3 (1:150; Abcam)/anti-CD8 (1:100; Abcam), anti-CD80 (1: 50; Abcam)/anti-CD86 (1:50; Abcam), and anti-CD19 (1:30; Abcam)/anti-CD20 (1:30; Abcam) antibodies at 4°C overnight. After the sections were washed with phosphate-buffered saline (PBS), they were incubated with Cy3-labeled goat anti-rabbit IgG antibodies (red) (1:1,000; KPL) and DyLight 488-labeled goat anti-mouse/rat IgG antibodies (green) (1:1,000; Abcam) as the secondary antibodies. Finally, the sections were incubated with 4′,6-diamidino-2-phenylindole (DAPI) to stain the nuclei (blue) and observed using a fluorescence microscope (Olympus).