Microscope image system

Immunohistochemical staining was performed on paraffin sections. Antigen retrieval was performed with antigen unmasking solution (Vector Laboratories). Endogenous peroxidase activity was quenched with 3% H₂O₂. After blocked with 5% normal serum, the slides were incubated with primary antibodies in a humidified chamber overnight. The next day, the slides were incubated with appropriate secondary antibodies and ABC solution using ABC Elite kit (Vector Laboratories). The immunoreactivity was revealed by incubating the slides in DAB solution. Nuclear staining was performed using hematoxylin. The slides were dehydrated, cleared, and mounted. The images were acquired and analyzed by NIS Element software with Nikon microscope image system (Nikon Instruments).

Immunohistochemistry was carried out on paraffin-embedded kidney sections [15 (link)]. Antigen unmasking solution (Vector Laboratories, Burlingame, CA, USA) was used to retrieve the antigen after fixation. Three percent H₂O₂ was used to block endogenous peroxidase activity before blocking. After incubation with 5% normal serum, slides were incubated with primary antibodies in a humidified chamber overnight. After washing, kidney sections were incubated with secondary antibodies, followed by ABC solution as previously reported [16 (link)]. Diaminobenzidine solution was applied to visualize the secondary antibody conjugated with horseradish peroxidase (HRP). Red substrate was used to visualize the secondary antibody conjugated with alkaline phosphatase (AP). Hematoxylin was used for nuclear staining. The sections were dehydrated with ethanol, cleared with histoclear, and mounted with mounting medium. NIS Element software (Nikon Instruments, Melville, NY, USA) with Nikon microscope image system (Nikon Instruments) were utilized to quantify the protein expression levels in the kidney.