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The gradient was as follows: starting at 10% B and increasing to 45% B over 2.7 min, then to 53% over 0.1 min, to 65% over 6.2 min, to 89% over 0.1 min, to 92% over 1.9 min and finally to 100% over 0.1 min

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After dehydration, the tissue was cleared in xylene for 15 min, followed by infiltration with soft and hard paraffin wax, and finally embedded in paraffin

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