Ma5 42523

Prior to staining, kidney sections from rats underwent deparaffinization in xylene, which clears the paraffin from the sections, followed by a graded rehydration process. This typically involves a series of alcohol baths of decreasing concentration to gently reintroduce water into the tissues. Then the rehydrated sections were then incubated with the following primary antibodies for targeted detection of proteins: GPX-4 (1:100, rabbit monoclonal, ab125066, Abcam), xCT (1:200, rabbit monoclonal, ab175186, Abcam), ACSL4 (1:200, rabbit monoclonal, MA5-42523, Invitrogen), and Nrf-2 (1:100, rabbit polyclonal, PA5-88084, Invitrogen). The primary antibody incubation was performed in a controlled environment at 4 °C overnight to allow for maximum binding specificity and efficiency. Following primary antibody incubation, the sections were washed to remove unbound antibodies, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody. Finally, the HRP-DAB system (Proteintech, Wuhan, China) was applied to visualize the sections, followed by staining with hematoxylin to detect HRP activity. All sections were imaged under an Olympus BX53F microscope.

Proteins were isolated from the collected cells and kidney samples using a radio-immunoprecipitation assay (RIPA) lysis buffer (R0010, Solarbio, Beijing, China). Following extraction, the protein levels were determined using a Bicinchoninic Acid (BCA) protein assay kit (PC0020, Solarbio, Beijing, China). The western blot analysis was conducted according to established methods. Primary antibodies utilized included GPX-4 (1:1000 dilution, ab125066, rabbit monoclonal, Abcam), xCT (1:1000 dilution, ab175186, rabbit monoclonal, Abcam), ACSL4 (1:1000 dilution, MA5-42523, rabbit monoclonal, Invitrogen), and Nrf-2 (1:1000 dilution, PA5-88084, rabbit polyclonal, Invitrogen). Secondary antibodies, used at a 1:5000 dilution, were acquired from Proteintech (Wuhan, China).