Deltavision elite microscope

For co-culture experiments images were taken every hour at different magnifications with an inverted fluorescence microscope (Leica AF6000). Cancer cell elongation was quantified manually by scoring individual cells using Image J “Cell Counter” plugin on taken images at 48 h co-culture. Elongated cell were defined as single cell with a minimal length-width rapport of 2:1, as well as at least one sharp-ending extremity. Cancer cells motility was quantified based on single cell track follow on 24 h microscope movies using Image J “Manual Tracking” and “Chemotaxis Tool” plugins.
Imaging of 2D and 3D spheroids assay were performed with 5× and 40× objectives with Leica AF6000 inverted fluorescence microscope. Invasion was quantified using Photoshop CS6 (Adobe) by calculating the invaded area on the 4 days image, normalized on initial spheroid size.
Western Blot images were quantified for protein level using Image J “Gel Analyze Tool” plugin.
For immunostaining images were taken using a DeltaVision Elite Microscope (GE Healthcare) with 63× and 100× objectives, followed by deconvolution treatment.
For immunohistochemistry images, slides were scanned using a NanoZoomer 2.0 HT (Hamamatsu) and images were extracted using NDP.view2 analysis software (Hamamatsu).

Colon sections were from previous animal studies using an AOM-induced CRC model in A/J mice [10 (link)]. Methcarn-fixed paraffin embedded colon sections were deparaffinized with xylene and rehydrated in an ethanol gradient. The slides were incubated in a citrate buffer at 95°C for 15 minutes, cooled to room temperature (RT), rinsed with PBS and incubated in a blocking buffer (PBS containing 1% Saponin and 20% BSA) for 30 minutes. The slides were then incubated with rabbit anti-ColVI (1:200, Abcam) at 4°C overnight, washed with PBS, and incubated with donkey-anti-rabbit Alexa 594 for 1 hour at RT. The slides were washed again, stained with DAPI, mounted and examined in a DeltaVision Elite microscope (GE Healthcare).

Immunofluorescence experiments for C. elegans were performed as previously described (Zhang et al., 2018 (link)). Images shown in Figure 4—figure supplement 2A were obtained from worms dissected and fixed in the absence of Tween-20 to retain soluble proteins. Primary antibodies were obtained from commercial sources or have been previously described, and were diluted as follows: Rabbit anti-RAD-51 (1:5000, Novus Biologicals, #29480002), Rabbit anti-pHIM-8/ZIMs (1:500, Kim et al., 2015 (link)), Goat anti-SYP-1 (1:300, Harper et al., 2011 (link)), Rabbit anti-SYP-2 (1::500, Colaiácovo et al., 2003 (link)), Chicken anti-HTP-3 (1:500, MacQueen et al., 2005 (link)), Mouse anti-HA (1:400, Thermo Fisher, #26183), Mouse anti-GFP (1:500, Millipore Sigma, #11814460001), Mouse anti-FLAG (1:500, Sigma, #F1804), anti-ALFA-At647N (1:500, Nanotag Biotechnologies, N1502-At647N). Secondary antibodies labeled with Alexa 488, Cy3, or Cy5 were purchased from Jackson ImmunoResearch (WestGrove, PA) and used at 1:500. All images were acquired as z-stacks through 8–12 µm depth at z-intervals of 0.2 µm using a DeltaVision Elite microscope (GE) with a ×100, 1.4 N.A. or ×60, 1.42 N.A. oil-immersion objective. Iterative 3D deconvolution, image projection, and colorization were carried out using the softWoRx package and Adobe Photoshop CC 2021.

DIC imaging was done using a Deltavision Elite Microscope (GE Healthcare) equipped with an sCMOS camera (PCO Edge 5.5 with a 6.5 µm pixel size), 0.55 NA long working distance condenser, and 20×0.75 NA air objective (Olympus). Both a diffuser and green filter (GE Healthcare) were used with the LED illumination.