Cytosol (200 μL) is incubated for 30 min at 4°C with different concentrations of oestradiol, tamoxifen and (4-Hydroxytamoxifen) or dihydroxytamoxifen administered in 10 μL methanol. Control tubes are incubated with 10 μL methanol alone and non-specific binding is determined in a parallel incubation of cytosol (200 μL) with methanol (10 μL) containing DES (5 × 10⁶ M). [2,4,6,7-³H]Oestradiol solution (50 μL) in TED buffer is added to each tube to give a final concentration of 2 × 10^-9 M. Incubation is continued for 4 h (4°C) and then 400 μL of a suspension of dextran-coated charcoal (250 mg % Norit A, 2.5 mg % dextran) in TED buffer are added and allowed to stand for 20 min. Tubes are centrifuged at 800 g for 10 min (4°C) and 400 μL samples of the supernatant are added to 10 mL tritium scintillator (6 g butyl PBD, 135 mL toluene, 720 ml dioxan, 100 g naphthalene, 45 mL absolute methanol). Samples are counted for 10 min in a liquid scintillation spectrometer. Counting efficiency is determined by external standardization (35-36 %). Results are represented as a percentage of the specifically bound radioactivity (c.p.m.) in the control tubes^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[3]
Animals of each sex are divided into two groups: one group receives 4-Hydroxytamoxifen [6 μg/0.1 mL sesame oil/day, subcutaneously (s.c.) starting at 06.00 h] injections for three consecutive days, while the other group receives an equivalent amount of sesame oil injection for 3 days. Four hours following the third injection, each group is then subdivided into two groups: one receives four cumulative doses of methamphetamine hydrochloride (10 mg/kg, s.c.), and the other receives a comparable volume of saline at 2-h intervals. Bilateral gonadectomy is performed under pentobarbital anesthesia (50 mg/kg, intraperitoneally). Five weeks after surgery,gonadectomized mice of each sex are randomly divided into six groups. Five groups of each sex receive three daily injections ofvarious concentrations of 4-Hydroxytamoxifen (0, 1.5, 3.0, 6.0, and 12.0 μg/0.1 mL sesame oil/day). Four hours following the third injection, mice receive four doses of methamphetamine (MA, 10 mg/kg) at 2-h intervals. The remaining group of each sex receives sesame oil pretreatment for three consecutive days, followed by saline injections, and serves as the control group^[3].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Jordan VC, et al. A monohydroxylated metabolite of tamoxifen with potent antioestrogenic activity. J Endocrinol. 1977 Nov;75(2):305-16.  [Content Brief]

  [2]. Davis KM, et al. Small molecule-triggered Cas9 protein with improved genome-editing specificity. Nat Chem Biol. 2015 May;11(5):316-8.  [Content Brief]

  [3]. Kuo YM, et al. 4-Hydroxytamoxifen attenuates methamphetamine-induced nigrostriatal dopaminergic toxicity in intact and gonadetomized mice. J Neurochem. 2003 Dec;87(6):1436-43.  [Content Brief]

  [4]. Zhang J, et al. Drug Inducible CRISPR/Cas Systems. Comput Struct Biotechnol J. 2019;17:1171-1177.  [Content Brief]