Fibreplug: ovarian follicles were transferred to the hook of the fibreplug which was vertically plunged in liquid nitrogen, held for 10 s and then placed buy Bortezomib into its pre-cooled plastic sleeves, sealed and stored for 20 min. Following storage in liquid nitrogen, fibreplugs were removed from the sleeves and rapidly immersed into a glass plate containing pre-warmed (28 °C) vitrification solution, where the ovarian follicles were released. Removal of CPAs was carried out in three steps, 2 min for each step. Immediately after warming, ovarian follicles membrane integrity was assessed by using trypan blue (TB) staining.
To carry out the TB assay, a 0.4% TB stock solution (Sigma–Aldrich, Dorset, UK) was diluted to 0.2% in 90% L-15 medium. Ovarian follicles were stained for 3 min Gefitinib solubility dmso with 0.2% TB solution at room temperature, and then washed three times in 90% L-15 medium. Those unstained were considered as membrane intact ovarian follicles, while the blue stained ones were considered as membrane damaged
follicles [24] and [46]. Total and membrane intact ovarian follicles counts were carried out under a light microscope. ATP content in the ovarian follicles was measured immediately after warming and 120 min later. For extract preparation the procedure described by Guan et al. [13] was employed. Briefly, two ovarian tissue fragments containing 30 stage III zebrafish ovarian follicles (15 follicles in each fragment) were added to 1 ml of an ice cold solution containing 0.5 M perchloric acid + 4 mM EDTA and homogenized with a conical glass pestle. The homogenate was centrifuged at 17,000g for 10 min at 4 °C in a refrigerated centrifuge. Supernatant was separated and neutralized with 2.5 M KOH to adjust the pH value to between 6 and GPX6 7. The neutralized supernatant was then centrifuged for 5 min
at 8000g and the new supernatant again collected. This extract was loaded into Eppendorf tubes and stored at −20 °C until the ATP determination. ATP released from follicles was measured using a commercial bioluminescence assay kit based on luciferin-luciferase reaction (FL-AA, Sigma–Aldrich, Dorset, UK) according to the manufacturer’s instructions. A luminometer (TD-20/20 – Turner Designs, Sunnyvale, CA, USA) was used for all measurements. Background light was measured and subtracted by running a blank containing deionised water. A seven-point standard calibration curve was routinely included in each assay. The ATP concentration was determined by the formula from the linear regression of the standard curve. Follicles from fresh control (kept in L-15 medium at room temperature) and vitrified groups were used in triplicates and assays were repeated three times on three different days.