Renal tissue specimens were fixed with Carnoy’s fixative in 10% methanol overnight. Fixed renal tissues were embedded in paraffin, and then cut at a thickness of 4 μm. The sections were mounted on adhesive glass slides (Matsunami Glass Ind., Kishiwada, Japan) and deparaffinized with lemosol (Wako Pure Chemicals, Osaka, Japan). The glomerulus histological change was assessed using PAS staining [48 (link)]. Mast cells were stained with 0.05% toluidine blue (Chroma-Gesellschaft, Stuttgart, Germany) [47 (link)].
Immunohistological examinations were performed using an anti-nephrin antibody (LS-B1382, LSBio, Seattle, WA, USA) and anti-MMCP-4 antibody (ab92368, Abcam, Cambridge, UK) according to a protocol described elsewhere [48 (link)]. In brief, to suppress endogenous peroxidase activity and nonspecific binding, the deparaffinized sections were incubated with 3% hydrogen peroxide and protein-blocking solution for 5 min at room temperature, respectively. Then, these sections were incubated with the above diluted primary antibodies overnight at 4 °C, followed by reaction with components from a labeled streptavidin-biotin peroxidase kit (Dako LSAB kit, Dako, Carpinteria, CA, USA) that included 3-amino-9-ethylcarbazole color development. Sections were then lightly counterstained with hematoxylin.
Immunohistological examinations were performed using an anti-nephrin antibody (LS-B1382, LSBio, Seattle, WA, USA) and anti-MMCP-4 antibody (ab92368, Abcam, Cambridge, UK) according to a protocol described elsewhere [48 (link)]. In brief, to suppress endogenous peroxidase activity and nonspecific binding, the deparaffinized sections were incubated with 3% hydrogen peroxide and protein-blocking solution for 5 min at room temperature, respectively. Then, these sections were incubated with the above diluted primary antibodies overnight at 4 °C, followed by reaction with components from a labeled streptavidin-biotin peroxidase kit (Dako LSAB kit, Dako, Carpinteria, CA, USA) that included 3-amino-9-ethylcarbazole color development. Sections were then lightly counterstained with hematoxylin.
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