Axio imager 2 microscope system

smRNA-FISH was performed as previously described (Dimitrova et al., 2014 (link)). Briefly, MEFs were seeded on coverslips and cultured for 24 hours in the presence or absence of etoposide before being fixed in 4% methanol-free formaldehyde in DEPC-treated water for 10 minutes at room temperature. After being washed twice in PBS, cells were dehydrated overnight in 70% ethanol at 4°C and stored for up to a week. Dehydrated coverslips were transferred to a hybridization chamber and equilibrated in Wash Buffer A for 5 minutes (LGC, Biosearch Technologies). Samples were incubated overnight at 30°C in hybridization buffer containing a 1:50 dilution of Stellaris probes conjugated to Quasar 570 or Quasar 670 dye. The following day, samples were washed twice with Wash Buffer A for 30 minutes at 30C, washed once with Wash Buffer B for 5 minutes at room temperature, and mounted in Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories). Samples were imaged using the Axio Imager 2 microscope system (Zeiss) with a PlanApo 633×1.4 oil DIC objective lens (Zeiss).

Lung tumorigenesis was initiated in cohorts of KC and KPC mice as described in (DuPage et al., 2009 (link)) by intratracheal infection with 1x10⁵ pfu UGPC lentiviruses . Mice were analyzed at 12 or 16 weeks post tumor initiation. For histological analyses, lungs were inflated with 4% paraformaldehyde, and fixed overnight in 4% paraformaldehyde, prior to dehydration in 70% ethanol. Fixed lungs were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Tumor burden scored as tumor area relative to total lung area was determined using the freehand selection tool and Measure feature in ImageJ on images acquired with an Axio Imager 2 microscope system (Zeiss) with a PlanApo 10x 0.3 objective lens (Zeiss). Tumor grade was scored as previously described (DuPage et al., 2009 (link); Nikitin et al., 2004 (link)).