Comparison between two cryopreservation techniques of human ovarian cortex: morphological aspects and the heat shock response (HSR)

Elevated Extracellular HSP72 and Blunted Heat Shock Response in Severe COVID-19 Patients

“…The difference between the concentration at 37 °C and 42 °C is used as an HSR index [ 19 ]. This test was applied previously, in several different conditions and diseases [ 17 , 32 ].…”

“…Cellular protein quantification was determined using a BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA), and samples (1 µg) were mixed with 5× Laemmli loading buffer [50 mM Tris, 10% SDS, 10% glycerol, 10% 2-mercaptoethanol, and 2 mg/mL bromophenol blue] at a final concentration of 1:5, boiled for 5 min, and then electrophoresed [ 32 ]. For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), equivalent amounts of protein (1 µg) were applied in a 10% polyacrylamide minigel for 2 h at 100V [ 32 ]. Proteins were then transferred onto nitrocellulose membranes (GE Healthcare, Chicago, IL, USA) according to the manufacturer’s instructions (Bio-Rad, Hercules, FL, USA) (2 h, 100 V) [ 32 ].…”

“…For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), equivalent amounts of protein (1 µg) were applied in a 10% polyacrylamide minigel for 2 h at 100V [ 32 ]. Proteins were then transferred onto nitrocellulose membranes (GE Healthcare, Chicago, IL, USA) according to the manufacturer’s instructions (Bio-Rad, Hercules, FL, USA) (2 h, 100 V) [ 32 ]. For immunoblotting, membranes were blocked in 2% BSA in a wash buffer [50 mM Tris, 5 mM EDTA, 150 mM NaCl (TEN)-Tween 20 0.1% solution, pH 7.4] for 30 min and then incubated overnight at a 1:1.000 dilution with monoclonal Anti-HSP70 antibodies that were produced from mice (Sigma-Aldrich, St. Louis, MO, USA) [ 32 ].…”

“…Proteins were then transferred onto nitrocellulose membranes (GE Healthcare, Chicago, IL, USA) according to the manufacturer’s instructions (Bio-Rad, Hercules, FL, USA) (2 h, 100 V) [ 32 ]. For immunoblotting, membranes were blocked in 2% BSA in a wash buffer [50 mM Tris, 5 mM EDTA, 150 mM NaCl (TEN)-Tween 20 0.1% solution, pH 7.4] for 30 min and then incubated overnight at a 1:1.000 dilution with monoclonal Anti-HSP70 antibodies that were produced from mice (Sigma-Aldrich, St. Louis, MO, USA) [ 32 ]. After appropriate washing, the membranes were probed with anti-mouse IgG and biotin antibodies at a 1:10.000 dilution (Sigma-Aldrich, St. Louis, MO, USA) for 1h.…”

Ovarian tissue bank for fertility preservation and anti-menopause hormone replacement

“…Galbinski et al also compared cryopreservation of ovarian tissue by slow freezing or vitrifying in a metal closed system, and found that the number of follicles in both groups was lower than that in the fresh group, but the intact follicles in the vitrification group were higher, and the heat shock protein 70 kDa response-HSR in the slow freezing group was higher. Hence, they believed that both methods could be used for cryopreservation of ovarian tissue (27).…”