Anti nephrin antibody

Mtdh and nephrin (podocyte marker) antibodies were used to investigate the location and expression of Mtdh in frozen renal tissue samples obtained from db/db and db/m mice. The slides were permeabilized with 0.05% Triton X-100 (Biosharp, Anhui, China) in PBS for 10 min and blocked with 5% goat serum mixed with 2.5% bovine serum albumin for 2 h. Afterward, these sections were incubated with anti-Mtdh antibody (1:100; Abcam) together with anti-nephrin antibody (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C overnight. Anti-Mtdh and anti-nephrin primary antibodies were detected using Alexa Fluor 546 donkey anti-rabbit and Alexa Fluor 488 goat anti-rabbit (1:1000; Invitrogen) secondary antibodies, respectively, and the samples were incubated with them for 6 h at 4 °C. Cell nuclei were stained with DAPI for 10 min before the observation of the samples under a light microscope (Nikon Corporation).

Kidney slides were deparaffinized and rehydrated followed by overnight incubation with an anti-nephrin antibody (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). Stained histological sections were examined with a Nikon 55i microscope at ×200 magnification with fluorescent excitation, and images were analyzed using Nikon NIS Elements Software (Nikon Instruments Inc., Melville, NY, USA). Positively stained areas specific for the target protein used were expressed as percent area relative to total area analyzed. Analyses were carried out by two observers blinded to sample identity.