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Clinical examinations, sample collection and analysis were carried out at 5 week intervals, from birth until one year old.
Several factors can account for these differences, including interobserver and intraobserver variations [ 29], gestational age at the examinations, sample size and the percentage of abnormal AFI in these studies.
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When macroscopically abnormal lungs were observed at post-mortem examinations, samples of lung tissue were obtained and submitted for histopathological examination, bacterial and fungal microscopy and culture, and detection of respiratory tract pathogens by PCR.
For histological examinations, samples were embedded in 30% ovalbumin, 0.5% gelatine and 1% gluteraldehyde in PBS.
An evaluation method of AFM tip shape using a probe examination sample and its evaluation criteria are stipulated in the Japanese Industrial Standard (JIS) R 1683: 2007 "Test method for surface roughness of ceramic thin films by atomic force microscopy".
Prior to examination, samples were platinum sputter-coated.
Prior to examination, samples were sputter-coated with platinum to prevent charging during FESEM imaging.
Prior to examination, samples were gold sputter-coated, to render them electrically conductive.
For histological examination, samples were fixed with 4% paraformaldehyde in a 0.1 M phosphate buffer (pH 7.4) for 3 days.
Before examination, samples were transferred back into LN2.
For SEM examination, samples were sputter coated with gold/palladium and observed with an FEI Quanta 600 (FEI Company, Hillsboro, OR, USA) scanning electron microscope at the Scripps Institution of Oceanography Unified Laboratory Facility.
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