Page 136 - Molecular features of low-grade developmental brain tumours

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After incubation with primary antibodies, sections were washed in PBS and stained with a polymer-based peroxidase immunohistochemistry detection kit according to manufacturer’s guidelines (PowerVision Peroxidase system, ImmunoVision, Brisbane, CA, USA). After washing, sections were stained with 3,3’-diaminobenzidin tetrahydrochloride ([50 mg Diaminobenzidine DAB], Sigma-Aldrich, Zwijndrecht, The Netherlands) and H2O2 in Tris-HCl (0.015% final concentration). The reaction was stopped by washing with dH2O, the sections were then counterstained with haematoxylin and dehydrated with ethanol (70%, 95%, 100%) and xylene (3x) and coverslipped.
Staining’s of MMPs were quantified by measuring the optical density (OD) of SEGA and control tissue using ImageJ. For each case, 2-3 representative photographs of periventricular white matter (n = 4) and when present nearby gray matter (n = 3) were taken (20x magnification). Three OD measurements were taken after haematoxyline – DAB colour deconvolution for each respective picture. Finally, the mean OD was calculated per case.
In situ hybridization
Paraffin slides from SEGA and autopsy controls (Table 1) were deparaffinated in xylene (3x) and rinsed in ethanol (2x 100%, 70%) and dH2O. Sections were pretreated using a pressure cooker in 0.1M sodium citrate buffer, pH 6.0, at 121°C for 10 min. The oligonucleotide probe for miR-320d (TCCTCUCAACCCAGCTUUT) contained LNA modification, 2-o-methyl modification and digoxygenin (DIG) label (RiboTask ApS, Odense, Denmark). Sections were incubated with the probe (1:750 dilution) in hybridization mix (600 mM NaCl, 10 mM HEPES, 1 mM EDTA, 5X Denhardts, 50% Formamide) for 1 hour at 56°C. Sections were washed with saline-sodium citrate (SSC) 2X for 2 minutes, SSC 0.5X for 2 minutes and SSC 0.2X for 1 minute (in agitation). After washing with sterile PBS, sections were blocked for 15 minutes with 0.02% Tween 20 and 1% NGS. Hybridization was detected with AP labelled with anti-DIG (Roche Applied Science, Basel, Switzerland). Nitro-blue tetrazolium chloride (NBT)/5-bromo- 4-chloro-3’-indolyphosphate p-toluidine salt (BCIP) was used as chromogenic substrate for AP (1:50 diluted in NTM-T buffer (100 mM Tris, pH 9.5; 100 mM NaCl; 50 mM MgCl2; 0.05% Tween 20). Expression of miR-320d was quantified by measuring the OD of SEGA and control tissue using ImageJ. For each case, 2-3 representative photographs of periventricular white matter (n = 9) and when present nearby gray matter (n = 7) were taken (20x magnification). Three OD measurements were taken for each respective picture. Finally, the mean OD was calculated per case.
Cell culture
As SEGA are thought to originate from neuroglial precursor cells that undergo an altered/ impaired development, we have chosen to use a glial cell type in a developmental state; (human fetal astrocyte-enriched cells) in order to study the role of miR-320d on MMP expression under basal conditions. Primary fetal astrocyte-enriched cell cultures were obtained from fetal brain tissue (n=3, 14–19 weeks of gestation) collected from medically induced abortions as described previously 47,48 with appropriate maternal written consent for brain autopsy.