After washing with distilled water, the sections were dehydrated through a series of 70, 90, 99, and 100% ethanol (1 min at each concentration), transferred to xylene for three washes of 1 min each, and then covered with coverslips and mounting medium (MP-500, Matsunami Glass Ind., Ltd., Osaka, Japan).
The cells were then fixed with paraformaldehyde (PFA) (4% final concentration), transferred to a 96-well glass bottom plate, and imaged in a Zeiss LSM 710 confocal microscope.
Equal amounts of cells were taken from each condition, treated with sodium azide (10 mM final concentration), transferred to a black, flat-bottom 96-well microtiter plate (Greiner Bio-One, Germany) and the RFP fluorescence was measured with a microplate reader (Synergy Mx, BioTek, Bad Friedrichshall, Germany).
After sterilization, 5 ml portions sterile mineral salt agar supplemented with sodium chloride to different concentrations were transferred to the wells of a six-well-plate.
Ten ml of MB solution of different initial concentrations was transferred to a series of 15-ml glass stoppered bottles, each containing 0.034 g of agar sample.
Analysis of blubber from premature and term sea lion pups and parturient sea lion cows demonstrated that high maternal organochlorine concentrations are transferred to the fetus (Greig et al. 2007).
After washing with distilled water the tissues were fixed in ammonium molybdate for 30 min, washed again, dehydrated in ethanol with increasing concentrations, and transferred to xylene overnight before mounting.
Then, the strain repeatedly cultivated in the low ethanol concentration was transferred to medium containing a high ethanol concentration and cultivated repeatedly in the same high-ethanol-concentration medium.
Initially phenol in water solution having concentration of 87%% was taken in the reactor, and then the formalin having 37%% concentration was transferred to the reactor.
