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EXPRESSION OF MICRORNAS MIR21, MIR146A, AND MIR155 IN TSC
USA; 1:2000), anti-human leukocyte antigen (HLA)-DP, DQ, DR (mouse anti-human leukocyte antigen (HLA)-DP, DQ, DR, mouse clone CR3/43; DAKO, Glostrup, Denmark; 1:100), and phospho-S6 ribosomal protein (Ser235/236; pS6, rabbit polyclonal, Cell Signaling Technology, Beverly, MA, USA; 1:50). For the detection of miRNA targets we used antibodies specific for IRAK1 (mouse clone 3F7, Sigma-Aldrich, St. Louis, Mo, USA; 1:300); TRAF6 (monoclonal rabbit, Abcam, Cambridge, MA, USA; 1:300); PDCD4 (poly- clonal rabbit, Abcam, Cambridge, MA, USA; 1:450); SHIP1 (polyclonal rabbit, Cell Signaling Technology, Beverly, MA, USA; 1:300); phosphatase and tensin homolog (PTEN; mono- clonal rabbit, Cell Signaling Technology, Beverly, MA, USA; 1:100).
All labeled tissue sections were evaluated by two independent observers blinded to clinical data for the presence or absence of various histopathological parameters and specific immunoreactivity (IR) for the different markers. We also semi-quantitatively evaluated the IR for different markers, such as GFAP, HLA-DR, TRAF6, IRAK1, PDCD4, SHIP1 and PTEN. The intensity of the staining was evaluated using a scale of 0-3 [0: -, no; 1: +/-, weak; 2: +, moderate; 3: ++, strong staining]. All areas of the tuber were examined and the score represents the predominant cell staining intensity found in each case. The frequency of GFAP, HLA-DR, TRAF6, IRAK1, PDCD4, SHIP1 and PTEN positive cells [(1) rare; (2) sparse; (3) high] was also evaluated to give information about the relative number of positive glial cells within the tumor. As described in previous studies 35, 39, the product of these two values (intensity and frequency scores) was taken to give the over- all score (total score; immunoreactivity score; IRS).
In situ hybridization
ISH for miR21, miR146a and miR155 was performed using 5’-3’ fluorescein (FAM) and dou- ble digoxygenin (DIG)-labeled Superior probes (Ribotask ApS, Odense, Denmark; see Supp. Table 1). The hybridizations were done on 5 μm sections of paraffin embedded materials as previously described 35. The probes were hybridized at 53oC (miR21) and 56oC (miR146a and miR155) for 1 h and the hybridization was detected with alkaline phospha- tase (AP) labeled anti-DIG (Roche Applied Science, Basel, Switzerland) and AP labeled anti-fluorescein (Roche Applied Science). NBT (nitro-blue tetrazolium chloride)/BCIP (5-bromo-4-chloro-3’-indolyphosphate p-toluidine salt) was used as chromogenic sub- strate for AP. Negative control assays were performed without probes (sections were blank). For double-staining, combining immunohistochemistry with ISH, sections were first processed for ISH and then processed for immunocytochemistry with GFAP, NeuN, (HLA)-DP, DQ, DR (HLA-DR) or pS6. Signal was detected using the chromogen 3-ami- no-9-ethylcarbazole (Sigma-Aldrich).
Cell cultures
Primary fetal astrocyte-enriched cell cultures were obtained from fetal brain tissue (14- 19 weeks of gestation) obtained from medically induced abortions with appropriate maternal written consent for brain autopsy. Tissue was obtained in accordance with the Declaration of Helsinki and the AMC Research Code provided by the Medical Ethics Committee of the AMC. Cell isolation was performed as described elsewhere 40, 41. Briefly, visible blood vessels were removed, after which the tissue was mechanically minced into smaller fragments. Tissue was enzymatically digested by incubating at 37˚C for 30
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