After being sacrificed, the knee joints of mice were collected and stained with HE staining. To quantitatively evaluate the severity of arthritis, a scoring system was employed referring to the reported protocol (Zhang et al., 2017 (link)). Histological changes were examined by microscopic evaluation and scored in a blinded manner by two independent observers.
The immunohistochemical analysis was performed as previously described with some modifications (Zhang et al., 2017 (link)). Knee joint sections on slides were incubated with anti-FOXO1 and anti-VEGF antibodies (Wanleibio, Shenyang, China). Subsequently, the sections were stained using the polymer HRP detection system (PV9001, ZSGB-BIO, Beijing, China) and were visualized with DAB Peroxidase Substrate Kit (ZLI-9017, ZSGB-BIO, Beijing, China). Following immunostaining, the synovium area in the joint of each section was evaluated under a microscope (LEICA DMi8) in three randomly selected areas at a magnification of 100×. Image-Pro Plus 6 (Media Cybernetics, Inc.) was used to analyze the average integrated optical density (IOD) according to a previously described protocol (Wang et al., 2019 (link)).
The immunohistochemical analysis was performed as previously described with some modifications (Zhang et al., 2017 (link)). Knee joint sections on slides were incubated with anti-FOXO1 and anti-VEGF antibodies (Wanleibio, Shenyang, China). Subsequently, the sections were stained using the polymer HRP detection system (PV9001, ZSGB-BIO, Beijing, China) and were visualized with DAB Peroxidase Substrate Kit (ZLI-9017, ZSGB-BIO, Beijing, China). Following immunostaining, the synovium area in the joint of each section was evaluated under a microscope (LEICA DMi8) in three randomly selected areas at a magnification of 100×. Image-Pro Plus 6 (Media Cybernetics, Inc.) was used to analyze the average integrated optical density (IOD) according to a previously described protocol (Wang et al., 2019 (link)).
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