Mouse testosterone elisa kit

Blood was collected from mice via the orbital vein and then centrifuged for serum collection. Testosterone concentrations in Etv5^−/− male and WT control littermates at 12 weeks of age were determined using the mouse testosterone ELISA kit (E05101m, CUSABIO, Wuhan, China). In brief, 50 μl of standards or samples were added to each well, with blank wells left empty. About 50 μl of HRP conjugate was added to each well except for the blank well. Subsequently, 50 μl of antibody was added to each well. The wells were mixed and then incubated for 1 h at 37°C. Contents from each well were aspirated and washed three times. The assay plate was blotted dry, and 50 μl of substrate A and 50 μl of substrate B were added to each well and incubated for 15 min at 37°C in the dark. After incubation, 50 μl of stop solution was added to each well, and the optical density (OD) of each well was recorded within 10 min using a microplate reader set to 450 nm.

We first randomly selected a female mouse to be included in the PCOS model group for pre-experimental use and then randomly divided the remaining 65 female mice into 5 groups with 13 females in each group. A group was randomly selected from the five groups as the control group. The other four groups (total 52 mice), along with the mouse that was randomly selected before (total 53 mice), were used for PCOS modeling. As a result, 66 female mice were randomly divided into model group (n = 53) and control group (n = 13). The model mice were given a 60% high-fat diet. They were subcutaneously injected with dehydroepiandrosterone (DHEA; Coolober technology Co., Ltd., Beijing, China; 0.6 mg kg⁻¹ day⁻¹, dissolved in 0.2 mL of soy oil) for 21 consecutive days. Mice in the control group were given a normal diet and subcutaneously injected with the same dose of 0.9% sodium chloride solution. On the first day after modeling, three mice in each group were randomly selected, blood was extracted after anesthesia, and serum-free testosterone was detected using Mouse testosterone ELISA Kit (CUSABIO, Wuhan, China). After the animals were killed, the ovaries were rapidly fixed in 4% paraformaldehyde solution. After being embedded in paraffin, they were cut into 4-μm-thick sections and stained with hematoxylin-eosin (H&E).