Giemsa stock solution

The evaluation of the menstrual cycle was implemented as defined in a previous study [15 (link)]. Briefly, vaginal smears were incubated in a 10% Giemsa solution (Giemsa stock solution, ROTH T862.1, Karlsruhe, Germany). The vaginal smears were classified into estrous (fertile phase), metestrous, diestrous, and proestrous phases. The regular cycle of mice lasts 4 days, and thus, no fertile phase within 4 days was defined as amenorrhea. The incidence of mice with a fertile phase in 4-day blocks is presented in Figure 1E and Figure 2E.

The estrous cycle was determined as previously described [11 (link)]. Vaginal smears were taken, fixated in methanol, and stained with 10% Giemsa solution (Giemsa stock solution, ROTH T862.1, TH.GEYER, Hamburg, Germany). The cell morphology of the vaginal smears was analyzed under a microscope using 20- and 40-fold objectives to stage the estrous cycle in estrous, metestrous, diestrous, and proestrous phases. The estrous phase is characterized by the occurrence of cornification and clustering of epithelial cells, whereas the other phases are characterized by the presence of leukocytes and nucleated epithelial cells (Supplementary Figure S1). Amenorrhea was defined as no detectable fertile phase (estrous phase) within the regular duration of the murine cycle, id est, 4 days. Therefore, the percentage of mice with an estrous phase in 4-day blocks was calculated.

Dulbecco’s modified Eagle’s medium (DMEM), Phosphate Buffered Saline (PBS), Penicillin/Streptomycin (PS), Glutamine, Insulin, Dexamethasone, sterile water, Gelatin B, AlCl₃ hexahydrate, sodium bicarbonate, HCl, KCl, PIPES, Methanol, Acetic acid, DPX mountant, Formaldehyde, DAPI, Tween 20, Triton X-100, Staurosporine, Ribonuclease A, Bovine Serum Albumin (BSA), MMS and Colcemid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tris Base, DMEM F12 Glutamax and Human recombinant Epidermal Growth Factor (EGF) were from Thermo Fisher Scientific (Waltham, MA, USA). FCS and Horse serum were from BioConcept Ltd (Paradiesrain, Allschwil, Switzerland). Lumogallion (4-Chloro-6-(2,4-dihydroxyphenyl-azo)-1-hydroxybenzene-2-sulfonic acid) was from Santa Cruz Biotechnology Inc (Dallas, TX, USA). DAKO Fluorescent mounting medium was from Agilent Technologies Inc (Santa Clara, CA, USA). Fluoromount G was from SouthernBiotech (Birmingham, AL, USA). Giemsa stock solution was purchased from Carl Roth (Karlsruhe, Germany).