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After removing the blocking solution, primary antibodies were added in optimized concentrations (see Table 1).
The phytohomones were added to the MS medium in optimized concentrations.
Immunohistochemistry was performed using an automated staining system (BenchMark XT, Roche-Diagnostics, Mannheim, Germany), with antibodies listed in Supplementary Table 1 in optimized concentrations and after adapted pre-treatment protocols for antigen retrieval.
Quantitative RT-PCR reactions were subsequently carried out using SYBR Green PCR master mix (Applied Biosystems) with primers and cDNA added in optimized concentrations, using a StepOne real-time PCR machine (Applied Biosystems) with cycling conditions at 95°C for 10 s, followed by 40 cycles of 55°C for 15 s, 60°C for 10 s.
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The Hela cells in optimized concentration were fast frozen in a cryo-plunger, and transferred to a dual beam SEM to be thinned with FIB milling.
Solutions of P1 and P2 in optimized concentration range of 0.14 0.35 mM were prepared from stock solution made of using 0.5 M HCl.
The appropriate antibody mixes or single stains in the optimized concentrations (diluted in FACS buffer) were then added onto the Fc blocked cells in additional 50 µl and incubated for 30 minutes on ice in dark conditions.
To combat their nutritional deficiencies, a modified poultry egg (MEΨ) was designed containing higher amounts of Cu, Mg and other antioxidants (vitamin E and linolenic acid) in their optimized concentrations.
Samples obtained from the three methods were analyzed by DLS at optimized concentrations in CH2Cl2.
Considering the onset potentials and Tafel slopes, we found that 2 and 2.3 mg/mL represent optimized concentrations of GO in terms of HER performance.
Parasites treated with optimized concentrations of each compound in single or double combination showed little effect on parasite growth.
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