Testosterone elisa kit

Serum samples were collected from the blood by “centrifugation at 2200 g for 15 min at 4°C and subjected to testosterone analysis using immunoassay ELISA Kit (Testosterone ELISA Kit, Abcam, Cambridge, UK)” [28 , 65 ]. The level of testosterone in each sample was calculated according to the manufacturer's instructions, as mentioned before [28 , 65 ].

Blood samples from PND 90 animals (5 CT males; 4 CT females; and 8 masculinized females) were collected (intracardiac puncture) after anesthesia with an intraperitoneal (ip) injection of ketamine (90 mg/kg; Nimatek) and xylazine (10 mg/kg; Ronpum 2%). Serum was isolated and testosterone levels measured using a testosterone ELISA Kit (Abcam, Cambridge, UK), according to the manufacturer’s instructions.

After 90 days, all animals were anesthetized again by the same technique described above for resection of surgical specimens and blood sample collection. The analysis of the duct deferens and measurement of the testicular size was standardized on the right side in all animals. Blood samples were collected from the venous plexus of the spermatic cord for serum testosterone dosage in duplicate (Testosterone ELISA Kit Abcam108666, USA). After sample collection, the animals were euthanized by a lethal dose of the anesthetics used.
The testicle was measured with a pachymeter in the longitudinal and transverse directions to calculate its size. Then, the right inguinal region was dissected in a block, and the duct deferens were isolated and sectioned in three axial segments, according to the relation with the mesh: 1) cranial: 1 cm above the mesh; 2) medial: in contact with the mesh; 3) caudal: 1 cm below the mesh. After the sample collection, the animals were submitted to euthanasia by means of a lethal dose of the anesthetic used.
The duct deferens segments were immediately fixed in 4% paraformaldehyde for at least 24 h. Following fixation, the samples were dehydrated, paraffin-embedded, serially sectioned at 5 μm, and mounted on glass microscope slides. Routine hematoxylin and eosin (HE) staining was performed for histological examination with light microscopy.

Serum samples were collected from each group. Measuring was conducted with the commercially available kit (mouse/rat testosterone ELISA Kit, CAT#: ab285350, Abcam, Cambridge, UK) (measuring range: 0.1–18 ng/mL). The specimens were diluted using the assay buffer included in the kit. By employing the provided testosterone standard (0 to 18 ng/mL), the standard curve was obtained. A total of 50 µL of each standard and specimen was placed into the corresponding wells of the microplate, which was coated with an antibody specific to testosterone. Additionally, 50 µL of the supplied testosterone-HRP conjugate was introduced to each well. The microplate was then sealed with the adhesive strip included in the kit and incubated for 2 h at room temperature on a shaker. Following the removal of the adhesive strip, the microplate was washed thrice with the kit’s wash buffer. Subsequently, the substrate was incubated for 15 min at room temperature in the absence of light, after which 50 µL of the stop solution was added to each well. The absorbance of every well was measured at 450 nm with a microplate reader. The testosterone concentrations of the specimens were determined using the standard curve, which was generated by plotting the absorbance values of the standards against their respective concentrations.

Blood samples were collected directly from the inferior vena cava using a 1-mL syringe at the end of the experiment. Serum was obtained by centrifugation at 2000× g for 10 min and stored at −70 °C until use. Serum LH levels were measured using a rat LH ELISA kit (Cusabio Biotech, Wuhan, China). Serum testosterone levels were measured using a testosterone ELISA kit (Abcam, Cambridge, UK). 17β-Estradiol (E2) levels were measured using an estradiol (rat) ELISA kit (BioVision, Mountain View, CA, USA). All kits were used according to the manufacturers’ instructions.

Upon completion of each experiment, blood samples were collected from the heart by direct puncture of the left ventricle using serum syringes. The samples were then centrifuged (GS-6R Centrifuge, Beckman Coulter, Fullerton, CA, USA) at 200× g and room temperature for 10 min, followed by serum storage at −20 °C. Testosterone concentrations in the serum were measured utilizing an ELISA reader (Victor3 ^TM X3, Perkin Elmer Inc., Waltham, MA, USA) and the corresponding enzyme immunoassay kit (Testosterone ELISA Kit; ab108666; Abcam Ltd., Cambridge, UK).