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.