Immuno-electron microscopy

Twelve weeks post xenografting, mice were euthanized by transcardial perfusion with Karnovsky’s fixative: 4% PFA (EMS 15700) in 0.1 M sodium cacodylate (EMS 12300), 2% glutaraldehyde (EMS 16000), p.H 7.4. For all xenograft analysis, transmission electron microscopy was performed in the tumour mass located in the CA1 region of the hippocampus. At room temperature the samples were post fixed in 1% osmium tetroxide (EMS 19100) for 1 h, washed 3 times with ultrafiltered water, before 2-h en bloc staining. The samples were dehydrated in graded ethanol (50%, 75% and 95%) for 15 min each at 4 °C before equilibrating to room temperature and washed in 100% ethanol twice, followed by a 15 min acetonitrile wash. Samples were immersed for 2 h in Embed-812 resin (EMS 14120) with 1:1 ratio of acetonitrile, followed by a 2:1 Embed-812:acetonitrile for 2 h, then in Embed-812 for 2 h. The samples were moved to TAAB capsules with fresh resin and kept at 65 °C overnight. Sections of 40 and 60 nm were cut on an Ultracut S (Leica) and mounted on 100-mes Ni grids (EMS FCF100-Ni). For immunohistochemistry, microetching was done with 10% periodic acid and eluting of osmium with 10% sodium metaperiodate for 15 min at room temperature on parafilm. Grids were rinsed with water three times, followed by 0.5 M glycine quench, and then incubated in blocking solution (0.5% BSA, 0.5% ovalbumin in PBST) at room temperature for 20 min. Primary rabbit anti-GFP (1:300; MBL International) was diluted in the same blocking solution and incubated overnight at 4 °C. The next day, grids were rinsed in PBS three times, and incubated in secondary antibody (1:10 10-nm gold-conjugated IgG TED Pella15732) for 1 h at room temperature and rinsed with PBST followed by water. For each staining set, samples that did not contain any GFP-expressing cells were stained simultaneously to control for any non-specific binding. Grids were contrast stained for 30 s in 3.5% uranyl acetate in 50% acetone followed by staining in 0.2% lead citrate for 90 s. Samples were imaged using a JEOL JEM-1400 TEM at 120 kV and images were collected using a Gatan Orius digital camera. Secondary antibody-only controls were used to compare for specific binding and quantification of images was performed by a blinded investigator.