Assessing LEP-R Expression in Iliac Bone

In the current study, the PV-9000 two-step immunohistochemical method was used to evaluate LEP-R level in slices of iliac bone tissue (22 (link)). Briefly, paraffin-embedded sections were deparaffinized and hydrated. Next, antigen retrieval was performed in 0.01 M citric acid (pH 6.0), and the sections were then incubated with a 3% H₂O₂ solution for 15 min at room temperature to block endogenous peroxidase activity. Subsequently, the sections were incubated with an anti-LEP-R antibody (cat. no. sc-8325; 1:150; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight in a humid environment, followed by incubation with a horseradish peroxidase-labeled secondary antibody (cat. no. PV-9000; 1:50; OriGene Technologies, Inc., Beijing, China) at room temperature for 20 min. Then chromogenic agents from the DAB chromogenic agent kit (Wuhan Boshide Biological Engineering Co., Ltd., Wuhan, China) were added to the sections. Finally, the sections were counterstained with Harris hematoxylin, dehydrated and sealed. Positive LEP-R expression was noted when the membrane presented brown-yellow staining. Sections were observed under a light microscope, and the integral optical density (IOD) of each field was measured with Image-ProPlus analysis software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA). The mean IOD value of five fields is reported as the IOD of the section.