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All these factors need to be considered

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Cells in chamber slides were fixed with 3 % formaldehyde in PBS at 4 C for 20 min and permeabilized with 0.5 % Triton buffer (0.5 % Triton X-100, 20 mM HEPES-KOH [pH 7.4], 50 mM NaCl, 3 mM MgCl) for 10 min at room temperature

Words like ice, cool, and frozen often indicate a cooling sensation, but thats not always the case

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The film, a distressing tale about a man caring for a child in a dystopian industrial setting, took five years to complete, finally appearing in 1977