Autodeblur

Specimens were imaged using an epifluorescence microscope (DM4000; Leica) equipped with DAPI, Endow GFP, and Texas red zero-pixel shift filter sets (Chroma Technology Corp.), a 63× 1.32 NA oil immersion objective, and a camera (CoolSNAP HQ; Roper Industries) controlled by MetaMorph 7. Z stacks were collected at 0.2 μm intervals and deconvolved using AutoDeblur (version X1; Media Cybernetics) for 10 iterations. Line scans (100 pixels wide) were performed using MetaMorph 7. For formalin-fixed, paraffin-embedded specimens stained as part of tissues microarrays, tiled images were collected on a DM4000 microscope equipped with Chroma filter sets (as above), a 20× HC PLAN APO NA 0.7 objective, CoolSNAP HQ2 grayscale CCD (for fluorescence) and Jenoptik ProgRes C10 Plus color CCD (for transmitted light) cameras, and a motorized xyz-stage (Ludl) controlled with custom MetaMorph 7 journals. Images were stitched using MetaMorph 7. Single images of H&E-stained slides were acquired using a MicroPublisher 5.0 CCD camera (Q Imaging) on a Leica DMLB microscope equipped with 10× NA0.25 and 40× NA0.65 HC FL PLAN objectives.

We captured images of labeled cells in the IM at the optical microscope using a CCD camera (Olympus DP70) attached to a microscope (Olympus optical microscope, BX 60) connected to a personal computer using a commercial imaging system (DPController). We captured labeled neuron profiles in the EM using a digital camera (ES1000W, Gatan, Pleasanton, CA, USA) at a magnification of 10,000–50,000X. Images were imported into Adobe Illustrator CS5 software (Adobe Systems Inc., San José, CA, USA) to assemble in figures. Fluorescent images were deconvolved using AutoDeblur (Media Cybernetics, Rockville, MD, USA) or Deconvolution Lab plugin for ImageJ. Minor adjustment of overall brightness and contrast were made but images were not retouched.