Dissection microscope

RT2-cancers were isolated from the pancreas of female RT2 or RT2.Stat1^−/− mice by intraductal injection of collagenase (1 mg ml⁻¹, Serva, Heidelberg, Germany)^{25 (link),31 (link)}. After injection, the pancreata were harvested, digested in collagenase solution at 37 °C for 10 min, and then mechanically disrupted. Whole encapsulated tumours were separated under a dissection microscope (Leica Microsystems) and further processed for immunofluorescence microscopy, immunohistochemistry or gene expression analysis. Alternatively, single tumour cells were obtained by incubation of the tumours in 0.05% trypsin/EDTA solution (Invitrogen) at 37 °C for 10 min. After incubation, RT2-cancer cells were seeded onto tissue culture plates.

Transwell migration and invasion assays were conducted using polycarbonate membrane 24-well Transwell inserts with 8 µm pore size (Costar, Corning™ Inc., Ottawa, ON, Canada). However, in the invasion assay, the transwell inserts were coated with 100 μL of Cultrex PathClear^® growth factor reduced, phenol red-free Matrigel (Trevigen Inc., Burlington, ON, Canada) diluted with serum-free RPMI 1640 medium into a final concentration of 0.15 mg/mL. Cells were transfected with NC, siRNA, miR-210-3p, or anti-miR-210-3p 24 h prior to migration and invasion assays. Cells were then collected using Accutase (Corning Inc.) and seeded on the top of the transwell insert at a density of 1.5 × 10⁴ cells for migration and 2.0 × 10⁴ cells for invasion per filter. Cells were seeded into the insert in serum free media while 10% FBS containing media was added outside the Transwell.
At 18 h post seeding into the transwell, cells were fixed and stained with Harleco Hemacolor Staining Kit (EMD Millipore, Oakville, ON, Canada). Non-migrated/invaded cells on the top of the membranes were wiped off using a cotton swab and membranes were cut with a blade and mounted on slides for quantification. Cells were visualized and photographed using a dissection microscope (Leica Microsystems, at 1.25×) and the numbers of cells migrated/invaded were counted using ImageJ as described previously [74 (link)].

Specimens were harvested as previously described (Figure 1)[23] (link). First, they were fixed in 10% neutral-buffered formalin overnight and then placed in alcoholic formalin for 2 hours, followed by 70%, 80%, 95% and 100% alcohol treatment. Next, specimens were placed in xylene 3 times, followed by 3 washed with liquid paraffin. Finally, specimens were embedded in rectangular paraffin blocks. The aneurysms were sectioned using an isomet low speed saw (SYJ-150 Kejing automation equipment co., Shenyang, China) at 1000 µm intervals in a coronal orientation, permitting long-axis sectioning of the aneurysm neck. Coil fragments were carefully removed with forceps under a dissection microscope (Leica Microsystems, Nussloch, Germany). The sections were then re-embedded in a paraffin block. A microtome with disposable blades sectioned the blocks at 5 mm intervals. Sections were floated in a water bath at 37°C and then mounted on superfrost plus slides and dried overnight in a 37°C oven. The samples were sectioned at 5 mm and Prussian blue staining was performed, as described previously.