Alexa fluor 594 conjugated goat anti rabbit

The renal tissue sections were deparaffinized and rehydrated in an alcohol gradient and xylene and then boiled in citrate buffer for 20 min for antigen retrieval. After blocking with 5% goat serum for 30 min, the sections were incubated with primary antibodies: anti-fibronectin (FN, ab2413, Abcam, Cambridge, UK) and anti-collagen-IV (col-IV, ab6586, Abcam) overnight at 4°C. After washing with phosphate-buffered saline (PBS), the sections were incubated with secondary antibodies (Alexa Fluor 594-conjugated goat anti-rabbit, Proteintech, Wuhan, China) for 30 min at 37°C in the dark. The images were captured by confocal laser-scanning microscopy (Nikon C2 Plus, Tokyo, Japan). The analysis of the overlay was performed by using ImageJ (National Institutes of Health, Bethesda, MA, USA).