14 mm microwell dishes

Immunostained sections were mounted in 14 mm microwell dishes (MatTek) and imaged using a Leica SP8 inverted 5 channel confocal microscope equipped with dual multiphoton (MP) lasers and a motorized stage. Microscope configuration was set up for three-dimensional analysis (x,y,z) of nematode sections. Mai Tai laser was tuned to 840 nm and InSight Deep See laser to 1150 nm excitation wavelengths. Second harmonic generation (SHG) signal was recorded at 420 nm wavelength. To collect tiled images of a whole section, z stacks consisting of 20 single planes (1 μm each, over a total tissue depth of 20 μm) were acquired and stitched automatically in LAS X (Leica Microsystems) post-acquisition. Images were processed using Imaris (Bitplane) software. Mean intensities of c-Abl fluorescence in various internal structures were analyzed using ImageJ (National Institutes of Health). Statistical analysis was performed using GraphPad Prism 7. Three sections per condition were analyzed.

Confocal microscopy of live tissue sections was performed for analysis of tissue architecture, cell segregation and cell numbers in mouse lung, kidney, brain, fat and liver ex vivo, at their physiological conditions. After euthanasia, mouse lungs were inflated with 1.5 % of low-melt agarose in RPMI at 37°C. Inflated tissues were kept on ice, in 1 % FBS in PBS, and sliced into 300–350 μm sections using Leica VT1000 S Vibrating Blade Microtome (Leica Microsystems). Mouse kidney, brain and liver were embedded in 1.5 % agarose in RPMI at 37°C prior to sectioning. Fat tissue was imaged directly. Tissue sections were stained with AF488 anti-mouse Laminin (NB300–144AF488, Novus Biologicals) for 2 h on ice. After staining, sections were washed 3 times and cultured in complete lymphocyte medium (Phenol Red-free RPMI supplemented with 10 % FBS, 25 mM HEPES, 50 μM b-ME, 1 % Pen/Strep/L-Glu and 1 % Sodium Pyruvate) in humidified incubator at 37°C. Sections were held down with tissue anchors (Warner Instruments) in 14 mm microwell dishes (MatTek), and imaged using DIVE microscope.