Positively charged microscope slides

The left lobes were fixed via intratracheal gravitational instillation of 10 ml of 10% buffered Formalin (Azer Scientific, Morgantown, PA) followed by immersion into the same fixative for at least 24 h at 4 °C. The tissue was cut into 3 mm sections at the apex, mid, and base portions (see schematic drawing in Fig. 6) followed by dehydration through an ethanol gradient with 30 min steps: 40, 60, 80, 90, 95, 100% 190-proof ethanol (Decon Laboratories), 100% 200-proof ethanol (Pharmco-AAPER, Kindermorgan, PA), and then cleared with two changes of 100% Substitute (Thermo) for 1 h each. These tissue sections were then embedded in molten paraffin (Fisher Scientific) at 60 °C, with 2 changes for 2 h each, and placed into blocks. Thin sections (5 μm thickness) were prepared using a Leica RM2255 microtome (Leica, Germany) and collected onto positively charged microscope slides (VWR, Radnor, PA). The samples were dried overnight and re-hydrated by two 10 min changes of Xylene Substitute, a 10 min change of Xylene/190-proof ethanol at 1:1 ratio, followed by 10 min changes of 100, 100, 90, 80, 70% 190-proof ethanol and, finally, deionized water. For assessing cell numbers, the slides were then mounted with DAPI-containing VectaShield (Vector Laboratories, Burlingame, CA), covered with coverslips and sealed with nail polish. Slides were kept at 4 °C until imaging.

Tumors extracted from mice were embedded in paraffin, cut on a microtome instrument (Leica) in 5 μm-thick sections that were then placed on positively charged microscope slides (VWR). Slides were de-paraffinized in xylene and rehydrated in a series of ethanol solutions (100%, 95%, 70%, 50%, water). Sections were permeabilized by incubating with PBS-T (PBS, 1% Triton X-100) for 15 min at RT and rinsed with water. Antigen retrieval was performed by incubating the slides for 13 min at RT with Proteinase K (at 20 μg/mL final concentration). Slides were washed with PBS 3 × 3 min and incubated with blocking buffer (5% (v/v) goat serum, 1% BSA, 0.5% Tween20 in PBS) for 60 min at RT. Slides were then incubated overnight at 4°C with a rat antibody against CD31 (at 1:100 dilution; Abcam) in a humid chamber. Slides were washed with PBS-T 3 × 10 min and with PBS for 5 min and incubated with the anti-rat secondary antibody conjugated with Cy3 fluorochrome (at 1:300 dilution; Jackson ImmunoResearch) for 2 h. After washing with PBS-T 3 × 10 min and PBS for 5 min, slides were mounted using VectaShield mounting medium with DAPI (Vector Laboratories).