0.5 m naoh

Cells were collected and then suspended in PBS (Sigma-Aldrich, USA). Carnoy’s Solution (3:1 methanol:acetic acid; Sigma-Aldrich, USA) was added in a 10:1 ratio with the sample suspension, then centrifuged for 5 min at 1500 rpm (300 g). The cell pellet was resuspended in Carnoy’s Solution, incubated for 15 min at –20 °C, and then centrifugation was repeated. Cold 10-μl drops were placed on pre-coated fluoro slides (Thermo Scientific, USA) and warmed to 52 °C. Slides were dehydrated, then decondensed in 0.5 M NaOH (Sigma-Aldrich, USA) solution for 1 min and then dehydration was repeated. Slides were then treated with 4 μl AneuVysion Multicolor DNA Probe (Vysis CEP 18/X/Y; Abbott Molecular, USA), and then incubated in a 37 °C humidified chamber for a minimum of 16 h. Following incubation, slides were rinsed in 0.3% NP 40 in 0.4× SSC warmed to 73 °C for 2 min followed by 0.1% NP 40 in 2× SSC for 10 s. Once dry, slides were counterstained with DAPI (Abbott Molecular, USA) and coverslipped. Slides were analyzed for diploidy by examining chromosomes X, Y, and 18 using DAPI, FITC, TxRed, and Aqua channels on an Olympus BX 61 fluorescence microscope and imaged using HCImage software (Hamamatsu Photonics, Japan). Human sperm was used as a positive control for analysis. Slides were stored between analyses and for the long-term at –20 °C.

DEAE-cellulose (10 g; Sigma Aldrich, Germany) dry gel washed with distilled water and left at 4°C for 16 h to remove small particles. The swollen gel was suspended in 0.5 M HCl (Merck Millipore, Germany) for 30 min and was filtered and washed with distilled water.The gel was suspended in 0.5 M NaOH (Merck Millipore, Germany) for 30 min (20 ) and washed with phosphate buffer (pH 6.3) 5×. The gel was packed into a XK 26/20 column (GE Healthcare, Sweden) with a 300 cm.h^-1 linear velocity (26.5 mL.min^-1) using preparative HPLC system (Waters, USA). The packed column was 85 mm bed height and 45 mL bed volume. The column was equilibrated by 3 column volume (CV), 0.07 M buffer phosphate pH 6.3 (19 ,21 (link)) at 225 cm.h^-1 linear velocity (20 mL.min^-1). Sample was loaded into the column at 150 cm.h^-1. Linear flow rate of 13.3 mL.min^-1 was applied to separate the adsorbed impurities.

Bovine skin was soaked in 0.5 M NaOH (Merck KGaA, Darmstadt, Germany), with ratio of skin/solution at 1:5 (w/v), for 3 days to remove non-collagenous proteins. The solution was changed every day. The alkaline treated skins were washed with tap water until neutral pH. The samples were then soaked in 0.1 M citric acid (Merck KGaA, Darmstadt, Germany) at a ratio of 1:5 (w/v) for 1 h. The swollen skin was mixed with distilled water at 1:5 (w/v) at (60 °C) for 6 h. The mixtures were then filtered using filter paper to remove insoluble materials. The supernatant was freeze-dried and subjected to analyses.