Paraffin wax

Conventional culture dishes that are 50 mm in diameter were purchased from Falcon (Sigma-Aldrich, Milan, Italy). Leibovitz’s L-15 medium, α-MEM GlutaMAX medium, insulin transferrin selenium (ITS) 100×, and LIVE/DEAD Fixable Far Red stain were purchased from Invitrogen (Milan, Italy). Penicillin-streptomycin 100×, amphotericin B 250 μg/mL, bovine serum albumin, L-ascorbic acid, L-glutamine 200 mM, Hoechst 33342, fructose, and α-thioglycerol were purchased from Sigma-Aldrich (Milan, Italy). Mayers’s hematoxylin and paraffin wax were purchased from Carlo Erba (Milan, Italy).

Twenty-eight fresh ovaries were harvested from abattoir bovines younger than 2 years at a local slaughterhouse (Ponte S. Giovanni, Italy), and were transported to the laboratory within 2 h from slaughter in Leibovitz’s L-15, 1% penicillin-streptomycin, 1 µg/mL amphotericin-B, at 4 °C. Ovaries were immersed in Leibovitz’s L-15, 2 mM glutamine, 3 mg/mL BSA, 1% penicillin-streptomycin, 1 µg/mL amphotericin B solution (hereafter referred to as handling medium), and fragments of ovarian cortical tissue about 1 mm thick, 5 mm wide, and 15 mm long were dissected with a scalpel, as shown in Figure 1. The actual size of exemplary fragments was verified by mechanical and histological means. Tissue was only harvested from smooth and regular regions of the ovary which presumably had never ovulated. Leibovitz’s L-15 medium was purchased from Invitrogen (Milan, Italy). Penicillin streptomycin 100X, amphotericin B 250 µg/mL, bovine serum albumin (BSA), L-glutamine 200 mM, and eosin-Y were purchased from Sigma Aldrich (Milan, Italy). Mayer’s hematoxylin and paraffin wax were purchased from Carlo Erba (Milan, Italy).