Dy07 microscope

The kidneys were immersed in 4% paraformaldehyde for 4 h and then transferred to 70% ethanol. Then the samples were cut into 4-μm-thick paraffin-embedded kidney sections and were incubated at 4 °C with AGEs (Abcam, UK) or RAGE (Abcam, UK) overnight. The sections were then incubated with a goat anti rabbit secondary antibody (Beijing Zhong Shan Golden Bridge Biotechnology Co., Ltd., China), for 1 h at room temperature. A negative control was included, in which the primary antibody was preincubated with a control peptide. Sections were examined using an Olympus DY07 microscope (Olympus, Tokyo, Japan) and digitized with a high-resolution camera (magnification 400×). Glomeruli or tubulointerstitial areas (N = 20, each) were randomly selected from each section and immunostaining was performed on the AGEs and RAGE. Immunostaining signals within each selected glomerular or tubulointerstitial area were highlighted and quantified using Image-Pro Plus quantitative software (Pax-it; Paxcam, Villa Park, IL, USA). Regions with positive staining were quantified and expressed as a percentage of the entire glomerulus.

The colons were fixed in 4% paraformaldehyde for four hours and then transferred to 70% ethanol for preservation. Subsequently, the samples were sliced into four-μm-thick sections and embedded in paraffin. These colon sections were subjected to overnight incubation at 4°C with specific primary antibodies: Recombinant anti-Claudin-1 antibody (GB15032, Servicebio, China) at a dilution of 1:500 (Mouse mAb), anti-Occludin Rabbit pAb (GB111401, Servicebio) at a dilution of 1:500, or anti-ZO-1 tight junction protein Rabbit pAb (GB11195, Servicebio) at a dilution of 1:500. Following this, the sections were treated with a goat anti-rabbit secondary antibody (Beijing Zhong Shan Golden Bridge Biotechnology Co., Ltd., China) for one hour at room temperature. The sections were examined using an Olympus DY07 microscope (Olympus, Japan) and high-resolution images were captured using a camera at a magnification of 400.