Hrp dab system

Kidney sections were fixed in 4% paraformaldehyde and stained using periodic acid-Schiff (PAS), hematoxylin–eosin (H&E), and Masson’s trichrome. We employed a microwave-based antigen retrieval technique to perform immunohistochemistry in 4-um paraffin kidney sections. The prepared tissue sections were incubated overnight at 4 °C with primary antibodies against FDX1 (1:100, 12592-1-ap, rabbit polyclonal, Proteintech, Wuhan, China), MAPK3 (1:100, A16686, rabbit polyclonal, ABclinal Technology, Wuhan, China), and NFKB1(1:100, A6667, rabbit polyclonal, ABclinal Technology, Wuhan, China). The sections were then washed and incubated with a secondary antibody at room temperature for 1 h. Finally, the sections were visualized using an HRP–DAB system (Proteintech, Wuhan, China) and stained with hematoxylin to detect HRP activity. The sections were observed under an Olympus BX53F microscope.

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.

Tibia tissues dissected from the mice were fixed with 10% neutral buffered formalin (NBF) for 72 h followed by decalcification in 14% EDTA (pH 7.4) for 3 weeks. Tissues were embedded in paraffin, and 5-μm sagittal-oriented sections were prepared for histological analyses with hematoxylin–eosin (H&E) staining and TRAP staining. Subsequently, osteoblast number, osteoclast number and surface, and adipocyte number and area were measured and calculated, respectively.
Immunohistochemical staining was performed as previously described [33 (link)], using primary anti-ALP antibody (Abcam, Cambridge, MA) at 4 °C overnight. Subsequently, an HRP-DAB system (Proteintech, Wuhan, China) was used to detect the immunoreactivity, followed by counterstaining with hematoxylin. The region of interest selected for histological and immunohistochemical staining analysis is 1 mm in length from 0.1 mm below the growth plate in proximal tibial metaphysis.