The calcium tracker calcein [10 mg/kg body weight (BW), Sigma Aldrich, U.S. No. C087-5G] alone or both calcein and tetracycline (30 mg/kg BW, Sigma Aldrich, U.S. No. T3383) were injected to 3-month-old female rats (n = 8) on days 8 and 7 before mating. Four rats were injected with calcein (once a day for 2 consecutive days) and four other rats were injected with calcein and tetracycline (once a day for 2 consecutive days). After confirmed mating with vaginal swab smear test, these rats were then permitted to gestation for 21 days followed by euthanasia to isolate their fetuses. Then we randomly selected two pups from each litter for sectioning. Fetal tibias were embedded into paraffin while longitudinal and transverse sections were examined under a Zeiss 1500 fluorescence microscope. We adopted the 555 nm excitation wavelength and collected signal at 603 nm emission wavelength for tetracycline (Macri-Pellizzeri et al., 2018 (link)); therefore, it appeared as red as shown in our figures. calcein exhibited green fluorescence under emission at 514 nm and excitation at 475 nm wavelengths.
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