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We present an improved technique using GMA, which permits both standardized embedding of 4×2×2 cm blocks and preparation of section series.
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In contrast to X-ray analyses, the determination of an optimum thickness through theoretical calculation is not straightforward, and an experimental approach, based on the preparation of sections of different thicknesses is usually more efficient.
Central or marginal tumor tissues were surgically excised for the preparation of sections.
Tissues from the 800 µg dose group were not examined due to the fact that the tumors were too small for the preparation of sections.
Specific real-time RT-PCR primers used to amplify were as follows: PC3 (rat sequence): 5'-GCGAGCAGAGACTCAAGGTT-3', 5'- CCAGTGGTGTTTGTAAT GATCG-3'; Id3: 5'-AGGAGTTTTTGCCACTGACC-3', 5'-CTCATCCATGC CCCTATA-bindingA-binding protein: 5'-GCTACTTGGGCGGCACTG-3', 5'-ACCAACAATCACCAGCAG CAG-3' Preparation of sections and hybridization were performed as reported previously [12].
Surgeons assisted pathologists to correctly identify resection margins during the preparation of sections in fixed specimens.
Preparation of sections was performed as described previously with slight modifications (Matsubayashi et al., 2008; Toda et al., 2008).
Paraffin embedding, preparation of sections and H&E stainings were performed as part of a histological staining service at the National Heart & Lung Institute.
To circumvent technical barriers and ensure that the tool is robust we have chosen to select features that were most reliable between a group of assessors and used standard, widely available, histopathological stains in preparation of sections.
To exclude autofluorescence secondary to the preparation of the sections, control sections were stained without primary antibodies.
To exclude artificial autofluorescence secondary to the preparation of the sections, control sections were stained without primary antibodies and no unspecific labeling was observed following incubation with secondary antibodies (data not shown).
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