40) objective. The criterion for having internalization was the presence in the neuronal soma of ten or more NK1R Everolimus order endosomes, defined as a small region of bright staining separated from the cell surface. The person counting the neurons was blinded to the treatment. All NK1R neurons in lamina I were counted in each histological section. In experiments in slices, at least three sections per slice were counted. In experiments in
vivo, four sections were counted per spinal segment. Confocal images were acquired using a Leica TCS-SP confocal microscope, using objectives of 20× (numerical aperture 0.70) and 100× (numerical aperture 1.40). One set of images (Fig. 1D) was acquired with a Zeiss LSM-710 confocal microscope using similar objectives. Excitation light for the Alexa Fluor 488 fluorophore
BIBF 1120 datasheet was provided by the 488-nm line of an argon laser. The emission window was 500–570 nm (emission peak for Alexa Fluor 488 is 519 nm). The pinhole was 1.0 Airy unit, corresponding to the objective used. Images were acquired in grayscale as confocal stacks of sections of 1024 × 1024 pixels. Photomultiplier gain and offset were individually adjusted for each image to avoid pixel saturation and loss of background detail. Each section was averaged 2–4 times to reduce noise. Images of the medial and central parts of the dorsal horn obtained with the 20× objective were used to show the location of the neurons imaged with the 100× objective (Fig. 1). Confocal stacks acquired with the 20× objective were processed using adaptive point spread function (‘blind’) deconvolution to reduce blur (Wallace et al., 2001; Cannell et al., 2006; Holmes et al., 2006), using the program autoquant X 2.0.1 (Media Cybernetics, Inc., Bethesda, MD, USA). Images taken with the 100× objective were not deconvolved because their native low blur made this unnecessary. The program imaris 6.1.5 (Bitplane AG, Zurich, Switzerland) was used to crop the confocal stacks in three dimensions. Images at 20× were cropped only in the z-dimension to choose the five brightest
optical Linifanib (ABT-869) sections. Images at 100× were cropped in x-y to show the soma and proximal dendrites of the target neurons, and in the z-dimension into three optical sections through the middle of the soma. Occasionally, several neurons were cropped from the same confocal stack. Image resolution was preserved in the cropping, so that pixels in Fig. 1 correspond to the pixels acquired by the confocal microscope. Voxel dimensions were 488 × 488 × 1180 nm with the 20× objective and 98 × 98 × 285 nm with the 100× objective. After cropping, a two-dimension projection picture was generated in Imaris and imported into adobe photoshop 5.5 (Adobe Systems Inc., Mountain View, CA, USA), which was used to make slight adjustments in the gamma of the images so that important details are clearly visible in Fig. 1. adobe photoshop was also used to compose the multi-panel figures and to add text and arrows.