2.7 Immunofluorescence analysis
Formalin formalin-fixed and paraffin-embedded kidney sections (4μm) were de-paraffinized, re-hydrated, and incubated with rodent declocker solution (Biocare Medical, Concord, CA, USA) at 950C for antigen retrieval. Kidney sections were then immunostained with anti-nephrin (1:100; Santa Cruz Biotechnology, Inc, Dallas, TX, USA) to determine renal expression of nephrin. Donkey anti-rabbit IgG H&L (Alexa Fluor® 488) secondary antibody (1:200; Abcam, Cambridge, MA, USA) was used for development with fluorescence quenching liquid (Vector Laboratories, Burlingame, CA, USA). Immunostained sections were examined by Nikon 55i fluorescence with a green excitation (200x magnification) and digital images were taken for analysis using Nikon NIS Elements Software. Nephrin expression in the kidney was determined by measuring the percentage of nephrin positive kidney areas. Two observers blinded to the identity of the samples conducted nephrin image analysis in kidney sections.