Q capture software

Tissue was fixed in zinc formalin, paraffin embedded, sectioned and stained with hematoxylin and eosin as previously described50 (link). Microscopic examination was performed by a board certified veterinary pathologist on two serial sections from 5–7 mice per group, focused on the following features: Numbers of inflammatory foci per lung lobe; numbers of lymphoid aggregates per lung lobe in perivascular, peribronchiolar, or intragranuloma locations; relative cellular composition (macrophages, lymphocytes, and neutrophils) of inflammatory foci; and presence or absence of necrosis (inflammatory cells or lung alveolar septae). Digital images were captured magnified 100 or 200 times normal using an Olympus BX41 light microscope fitted with a U-TVO camera and Q-Capture software. The images were minimally processed using Adobe Photoshop CS5.1 to increase brightness and contrast by 10%, and adjust resolution to 300dpi.

Immunohistochemistry was performed as previously described (32 (link)). Briefly, paraffin embedded tissue slides with human HCC tissue microarray (TMA) (NBP2-30221, Novus Biologicals) were deparaffinized and rehydrated, endogenous peroxidise activity was blocked with 3% hydrogen peroxide, antigen retrieval was performed in 10 mmol/L citrate buffer, and nonspecific binding was blocked with blocking reagent. HAVCR2 (ab185703, Abcam) and C10ORF54 (CL3975, Invitrogen) antibodies were applied at 1:300 and 1:20 concentrations, respectively. Slides were incubated overnight at 4°C, followed by 30 min incubation with secondary anti-mouse or rabbit antibody HRP (Dako). The chromogen used was 3-amino-9-ethylcarbazole. Human normal and cancerous lung tissue was used as the positive control for both the antibodies and a negative control, for which the primary antibodies were substituted with the same concentration of mouse or rabbit IgG. Images were captured using a Olympus CX41 microscope and QCapture software. Immunohistochemical reactivity was evaluated by two independent investigators. The expression of HAVCR2 and C10ORF54 were categorized into positive staining or no staining.

Matrigel (Life Technologies, Burlington, ON, Canada) was diluted 1:6 in DMEM (Life Technologies, Burlington, ON, Canada) and 20 μl of diluted Matrigel was plated on to the upper compartment of a Falcon cell culture insert (cat #353097; BD Bioscience, Mississauga, ON, Canada). Approximately 1x10⁵ cells were cultured in 200 ul of serum-free media in the upper compartment of the insert. The bottom well was filled with 300 ul of media containing serum. Cells were cultured at 37 °C and 5 % carbon dioxide for 20 h. Media was then aspirated from the lower chamber and the bottom of the insert was fixed with 5 % glutaraldehyde in 1xPBS, for 10 min, washed with water and stained with 0.5 % toluidine blue staining solution 10-20 min at room temperature. The inner surface of the upper chamber was then wiped clean and cells that had migrated to the bottom of the insert were visualized using an Olympus IX71 inverted microscope (Toronto, ON, Canada) and Q-capture software (Surrey, BC, Canada). The number of cells on the bottom of the insert were counted manually.