Methods
Ethics Statement
Anonymized human brain tumor (GBM) tissue samples and basic clinico-pathologic data were obtained through the Clark Smith Brain Tumour and Tissue Bank at the University of Calgary and Calgary Laboratory Services, Calgary, AB (ethics approved for biobanking and previous patient consent granted at time of banking). Tissue was used according to the policies of the institutional review boards of Calgary Laboratory Services and the Calgary Health Region Ethics Board. Further ethics review and approval for this study (ID# E-23011) was provided by the Conjoint Health Research Ethics Board (University of Calgary, Calgary, AB).
GBM Tissue Samples
All samples were part of routine clinical care for diagnostic and treatment purposes and were designated as excess material by the consulting and consenting neuropathologist. Hematoxylin and eosin stained formalin-fixed paraffin-embedded sections were reviewed by a neuropathologist for confirmation of a diagnosis of high-grade glioma (glioblastoma WHO grade IV) as per World Health Organization criteria.[18]
Molecular Characterization of GBM Tumors
Molecular characterization of IDH and EGFR was performed by the clinical Molecular Diagnostics Laboratory at Calgary Laboratory Services on formalin-fixed paraffin-embedded (FFPE) sections. Briefly, IDH1 and IDH2 mutational analyses were performed using a multiplexed SNaPshot® reaction and detection by capillary electrophoresis.[25] Analysis of EGFR amplification was performed by EGFR colorimetric in situ hybridization using standard methods with the EGFR probe #84-1300 (Zymed Laboratories), and scored by a neuropathologist as follows: 'amplified EGFR' indicates >10 signals/nucleus in >80% of tumor cells and 'not amplified' indicates 2 signals/nucleus in tumor cells.
Antigen Retrieval Method (ARM)
Slides containing the FFPE tissue sections were deparaffinised in xylene and passed through a graded ethanol series, rinsed with cold tap water and transferred to a Coplin jar on a hot plate containing a 100°C Tris-EDTA-Tween (w/v) solution (0.121% Tris HCl, 0.0379% EDTA, 0.05% Tween-20, pH adjusted to 9.0). The sections were boiled for 60 minutes and then allowed to reach room temperature while remaining in the same solution. The slides were washed in phosphate buffered saline (PBS) for 10 minutes and processed for IIF.
Indirect Immunofluorescence (IIF)
Formalin-fixed paraffin embedded tissue sections were treated with the above ARM (section 3.4.). Cells were blocked in 10% normal goat serum (NGS; Antibodies Incorporated, Davis, CA) and 2% bovine serum albumin (BSA; Sigma-Aldrich) for 30 minutes at room temperature (RT) and incubated with primary antibodies at appropriate working dilutions overnight at 4°C. Primary cilia were marked by mouse anti-acetylated tubulin at 1:100 dilution (Sigma, St. Louis, MO). After washing with PBS, cells were incubated for 2 hours in a dark chamber with Alexa Fluor (AF) 488 (green) secondary goat fluorochrome-conjugated antibodies at 1:100 dilution (Invitrogen). Slides were washed in several changes of PBS, cell nuclei counterstained with 4',6-diamidino-2-phenylindole (DAPI), mounted in Vectashield (Vector Laboratories, Burlingame, CA) and examined for IIF using a 100x objective on a Leica DMRE microscope equipped with epifluorescence and an Optronics camera. Appropriate IIF controls with no primary antibody revealed no detectable bleed-through between microscope filter sets.
Electron Microscopy (EM)
Fresh GBM samples were immersed in a fixative containing 3% glutaraldehyde in Millonig's phosphate buffer and stored at 4°C for 48 hours. Samples were immersed post-fixation in 2% OsO4 for 20 minutes and then dehydrated in ethanol and infiltrated with Polybed 812 resin (Polysciences Inc., Warrington, PA). Polymerization was performed at 37°C for 24 hours. Silver-gray sections were cut with an ultramicrotome (Leica) equipped with a diamond knife, stained with uranyl acetate and lead citrate and then examined in a H-700 Hitachi electron microscope. For each sample, 10 grids were examined on standard sections. Approximately 500 cells were examined in each tissue sample.
BMC Clin Pathol. 2014;14(40) © 2014 BioMed Central, Ltd.
