Ratlaps enzyme immunoassay kit

Five-week-old female BALB/c nude mice were randomly divided into 5 groups, with 7 mice per group. Mice were anesthetized with a Zoletil (30 mg/kg)/Rompun (10 mg/kg) mixture. MDA-MB-231 cells (1 × 10⁶ cells/50 µL HBSS) were injected into the medullary cavity of the proximal tibia of the mice. Control mice received HBSS alone. After twenty-four hours, chemerin at the indicated doses in 0.1% BSA was intraperitoneally injected every two days for 5 weeks. On day 35, the collected tibiae were scanned with a μCT system (SkyScan 1076, SkyScan, Aartselaar, Belgium). Three-dimensional (3D) images were generated using NRecon software (SkyScan). Serum levels of calcium, CTX, and TRAP 5b were determined using a QuantiChrome calcium assay kit (BioAssay Systems, Hayward, CA, USA), a mouse TRAP assay kit (Immuno Diagnostic Systems, Boldon, UK), and a RatLaps enzyme immunoassay kit (Immuno Diagnostic Systems), respectively.

PGE2 concentrations in the serum and bone marrow were determined by PGE2 ELISA kit (Cayman Chemical, 514010) according to the manufacturer’s protocol. Mice serum was collected as described above. We also performed osteocalcin and CTX ELISA of serum using a mouse osteocalcin enzyme immunoassay kit (Biomedical Technologies, BT-470) and a RatLaps enzyme immunoassay kit (Immunodiagnostic Systems, AC06F1).
Western blot analysis was conducted on the basis of the protein lysates from the hypothalamus of mice or cultured cell line. The lysates were centrifuged; the supernatants were collected and separated by SDS-PAGE PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and then blotted on the polyvinylidene fluoride membrane (Bio-Rad Laboratories). Specific antibodies were applied for incubation, and the proteins were detected by using an enhanced chemiluminescence kit (Amersham Bioscience, RNP2108). The antibodies used for western blotting were HTR2C (Abcam, ab197776, 1:500), CREB (Cell Signaling Technology, 9197, 1:500), p-CREB (Abcam, ab32096, 1:500), COX2 (Abcam, ab15191, 1:1000), and GAPDH (Cell Signaling Technology, 5174, 1:1000).