Confocal microscopy, image processing, and quantification of Y1R co-localization with INs

Representative confocal images of Y1R co-labeling with markers of excitatory or inhibitory interneurons were acquired with a Leica ABOS TCS SP5 inverted laser scanning confocal microscope, fitted with a 100x oil immersion objective (numerical aperture 1.46). The microscope is a Leica DMI 6000 with LAS AF 2.7.2.9586 software. Laser excitation lines and emission windows for the different fluorophores were: Alexa Fluor 488 - excitation 488 nm (Ar laser), emission 505–555 nm; Alexa Fluor 568 - excitation 543 nm (diode laser), emission 565–615 nm; DAPI - excitation 405 nm (HeNe laser), emission 435–485 nm. Line averaging was used to decrease signal to noise ratio. Adobe Illustrator CS6 (Adobe Systems Inc., Mountain View, CA) was used to assemble the multi-panel figures.

Quantification of staining in randomly-selected sections was performed with NIS Elements Advanced Research software. To distinguish immunohistochemical staining patterns of tibial and sural terminals, we selected ROIs spanning the medial-central and central-lateral regions, respectively, of lamina II of the tibial and sural across the mediolateral axis of the dorsal horn. Only DAPI-labeled cells were counted. Three animals per group and three slices per animal were quantified for Y1R and/or calbindin, calretinin, PKCγ, or Pax-2.