Data Management And Sharing System of China National Clinical Research Center for Neurological Diseases

Journal：Brain Tumor Pathology

Publication type: Research Article

IF(IF): 3.298

PMID: 27307151 Link To Pubmed Full text Download

The chromosome 19q13.42 status was examined using fluorescence in situ hybridization (FISH) with a 19q13.42/ CEP19 dual color probe. Briefly, 4-mm-thick FFPE tissue sections were dewaxed, incubated for 20 min at 95 C in a heat pretreatment solution, and digested with an enzyme reagent at 37 C for 15–25 min. After dehydration, 10 lL of a probe mixture that contained 1 lL of a 19q13.42/CEP19 probe (Healthchart LLC, Memphis, TN, USA), 2 lL of purified H2O, and 7 lL of LSI hybridization buffer (Healthchart LLC) were applied to each target area; the slides were subsequently covered with glass coverslips and sealed with rubber cement. Co-denaturation and hybridization were conducted using a Hybridizer device (Dako, Glostrup, Denmark) programmed for co-denaturation (5 min at 95 C) and overnight hybridization (16 h at 37 C). After removing the coverslips, slides were washed and dried in the dark, and hybridization was visualized by applying 15 lL of DAPI/VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA, USA). Signals were scored in at least 100 non-overlapping intact cells. The evaluated loci were 19q13.42 (RP11-381E3 and RP11-634C1, labeled red) and CEP19 (centromeric DNA, labeled green). Specimens were considered amplified for the 19q13.42 locus when the target/reference ratio was[2.0 or tight clusters of 19q13.42 locus probe signals were observed.