4-hydroxytamoxifen preparation
4-Hydroxytamoxifen (4-OHT; Sigma, Cat# H6278) was dissolved completely at 20 mg/mL in 100% ethanol on a shaker at 37°C, which required about 15 min. The solution was then stored in 250 µL sealed aliquots at -20°C for up to several weeks. On the day of use, an aliquot of 4-OHT in ethanol was redissolved on a shaker at 37°C for about 15 min, and castor oil:sunflower seed oil mixture (1:4) (Sigma, Cat #s 259853 and S5007) was added at a volume ratio of 1:2 (4-OHT: 6.7 mg/mL). The mixture was vortexed thoroughly for 1 min until the 2 phases were indistinguishable. The ethanol was then evaporated off via vacuum centrifugation to give a final concentration of 10 mg/mL 4-OHT. This was light protected and stored at 4°C until 30 min before injection, which occurred within 5-6 h of preparation. Based on in-house pilot tests and relevant literature, 4-OHT was injected i.p. at 50 mg/kg .