W1 spinning disk confocal microscope

For quantifying epidermal fibers from the GFP-stained hind paw skin sections, Z stack images were captured using an inverted epifluorescence microscope (Axiovert 200M microscope; ZEISS, Jena, Germany) with Apotome. Three images per tissue section, five sections per hind paw (a total of 15 images from one hind paw) were examined. The number of epidermal fibers passing through the epidermis-dermis junction (under the nuclei of basal epithelial cells identified by DAPI staining) were counted in each image, and values from 15 images were averaged.
The whole-mount stained skin was imaged using a Nikon W1 Spinning Disk confocal microscope at X10 objective. Consecutive Z-stack images were captured at 0.5 μm depth intervals from the selected skin spots until the fluorescence signal became undetectable. The Z projection was applied to a stack of all imaging sections, and single merged imaging of the skin spot was created. The raw images were processed to remove nonspecific fluorescence in the skin. All of the images were quantified and analyzed with ImageJ software. For all images, the background grayscale was eliminated, and binary images were generated. CGRP+ area and the entire region of interest (ROI) area were measured, and the ratio of the CGRP+ area/ROI area was calculated.

Dissections and staining were carried out as previously described (Flaherty et al., 2010 (link)). Briefly, testes were dissected in 1x phosphate buffered saline (PBS), fixed for 15 minutes in 4% paraformaldehyde (PFA) in 1xPBS, washed for 1 hour at 25°C in 1xPBS with 0.5% Triton X-100, and blocked in PBTB (1xPBS 0.2% Triton X-100 and 1% bovine serum albumin) for 1 hour at 25°C. Primary antibodies were incubated overnight at 4°C except for pSTAT which was incubated overnight at 25°C. They were washed two times for 30 minutes in PBTB and incubated for 2 hours in secondary antibody in PBTB at 25°C and then washed two times for 30 minute in 1xPBS with 0.2% Triton X-100. They were mounted in Vectashield or Vectashield with DAPI (Vector Laboratories). For 5-ethynyl-20-deoxyuridine (EdU, Life Technologies) labeling, samples were incubated for 30 minutes before fixation in Ringer’s medium containing 10 μM EdU. Testes were fixed and processed normally for antibody labeling and then treated per manufacturer’s instructions. Confocal images were captured using Zeiss LSM 510 and LSM 700 microscopes with a 63x objective. Z-stacks for 2D and 3D images were captured on a Nikon W1 spinning disk confocal microscope with lasers at 405, 488, 561, 640 nm, narrow pass filters for emission, a SR HP Plan Apo 100X 1.35 Silicon Oil λS DIC lens, and an Andor 888 Live EMCCD camera.