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At the end of each agitating period, the slumps of the mixtures were restored to the initial slumps using a superplasticizer and specimens were thereafter prepared for strength measurement.
These specimens were thereafter thoroughly washed in Tyrode's solution containing 10% sucrose at 4°C overnight, mounted on thin cardboard in OCT embedding medium (Miles Laboratories, Naperville, Ill ., and frozen in propane chilled with liquid nitrogen.
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The tissues were thereafter, according to routine standard methodology for pathological specimens, embedded in paraffin until histopathological examination.
Blood specimens were centrifuged immediately thereafter; and shipped frozen in dry ice to central clinical laboratory (certified by ISO 15189 and ISO 17025) in Tao-Yuan General Hospital.
For cell culture purposes, the biopsy specimens were minced and thereafter maintained in RPMI 1640 (Life Technologies, Grand Island, NY, USA) supplemented with 10% foetal calf serum (Life Technologies), 5 m M L-glutamine, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin at 37°C in a humidified 5% CO2 atmosphere.
Thereafter, specimens were embedded into paraffin and cut to 5-μm-thick sections.
Thereafter, specimens were subjected to cyclic and continuous chloride attack for defined time periods.
Thereafter, specimens were processed in an automated embedding system, sectioned at 4 6 microns and finally stained with haematoxyline and eosin.
Thereafter, specimens were rinsed in PBS (without NaN3) and mounted in Fluoromount-G with DAPI (SOUTHERN BIOTECHNOLOGY ASSOCIATES, Inc., Alabama, USA) or Vectashield with DAPI (VECTOR LABORATORIES, Burlingame, USA).
Volunteers remained in the study ward for the first 12 h of each phase; thereafter specimens were collected at outpatient visits.
Thereafter, specimens were washed 3 × 5 min in PBS and incubated with the DAB substrate kit (Vector Laboratories), according to the manufacturer's instructions.
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