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 .