Slowfade light antifade

For immunofluorescence staining, the sections were deparaffinized in xylene, rehydrated in graded ethanol (99–70%), and boiled in TEG-buffer to improve target availability. To prevent non-specific binding, tissue sections were blocked with 50 mM NH₄Cl in PBS and subsequently incubated in PBS with 1% BSA, 0.2% gelatin and 0.05% saponin. Next, slides were incubated overnight with KIM-1 antibody (R&D Systems) diluted in PBS containing 0.1% BSA and 0.3% Triton X-100 at 4 °C. After incubation, slides were washed with PBS containing 0.1% BSA, 0.2% gelatin and 0.05% saponin, and subsequently incubated with a secondary antibody (Alexa Fluor 568) and counterstained with DAPI (Sigma). Slides were mounted using Slowfade Light Antifade (Invitrogen, Carlsbad, CA, USA). Sequential overlapping images were taken and merged in Photoshop© CS5.
In order to evaluate fluorescence intensity, 10 pictures (20× magnification) distributed throughout the whole kidney were used. The threshold was set using a positive and negative control, and ImageJ software was used to calculate the intensity of KIM-1 immunolabeling.

For immunolabeling, the sections were deparaffinized overnight in xylene, rehydrated in 99% to 70% ethanol, and boiled in TEG-buffer to improve target availability. Non-specific binding to free aldehyde groups was blocked with 50 mM NH₄Cl in PBS followed by incubation in PBS containing 1% BSA, 0.2% gelatin, and 0.05% saponin. The tissue slides were incubated with primary antibody KIM-1 (R&D Systems) diluted in PBS containing 0.1% BSA and 0.3% Triton X-100, in a humidified chamber overnight at 4 °C. After washing in 0.1% BSA, 0.2% gelatin, 0.05% saponin in PBS, the tissue slides were incubated with Alexa fluor 568 secondary antibody and DAPI (Sigma) for nuclear staining. Tissue slides were mounted using Slowfade Light Antifade (Invitrogen, USA). Images were taken to overlap and merged in Photoshop© CS5.