LIN28A, a polyclonal antibody (A177, #3978, Cell Signaling Inc, Boston, MA, USA). IHC was
performed with an automated stainer (Benchmark; Ventana XT) using antigen-retrieval protocol
CC1 and a working antibody dilution of 1:100 with incubation at 37 °C for 32 min
(see supplementary material) page 3 line 5
Fluorescence in situ hybridization
Two-color interphase FISH was performed on de-paraffinized sections using two custom-made
fluorochrome- labeled locus-specific probes: FITC-labeled probe 634C1 corresponding to locus
19q13.42 and digoxigenin-labeled probe 2659N19 corresponding to locus 19p13 as a reference.
Pretreatment of slides, hybridization, post-hybridization processing, and signal detection were
performed as previously described (1). Samples showing sufficient FISH efficiency (90% nuclei
with signals) were evaluated by two independent investigators. Signals were scored in at least 200
non-overlapping, intact nuclei. Metaphase FISH for verifying clone mapping position was
performed using peripheral blood cell cultures of healthy donors as previously outlined (2).
Specimens were considered amplified for the 19q13.42 locus when more than 10% of tumor cells
exhibited either more than eight signals of the corresponding probe with a reference/control ratio
2.0 or innumerable tight clusters of signals of the reference locus probe. Polysomy 19 was defined
as 10% of nuclei containing three or more signals for both the target and the reference probes
according to the sensitivity of the probe tested in control brain tissue
(see supplementary material) page 3 line 13
Copy number aberrations (CNAs)
Copy number aberrations (CNAs) using data generated with Illumina Human Methylation 450 k
BeadChip arrays as described previously described (3). For the detection of amplifications,
chromosomal gains and losses, automatic scoring was verified by manual assess- ment of the
respective loci for each individual profile as previously described (4).
(see supplementary material) page 3 line 17
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metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum Genet
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