Ix81 microscope

Fresh frozen muscle or sciatic nerve tissue was cryosectioned (10 μm) and fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) (4% PFA/PBS) or stained nonfixed. Sections were incubated in blocking solutions (5% donkey serum and 0.3% Triton-X in PBS) for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies, diluted in 5% donkey serum in PBS. Sections were washed three times with PBS and incubated at room temperature for 1 h with the appropriate secondary antibodies. Images were acquired with an Olympus iX81 microscope, a Zeiss Axio Scan.Z1 Slide Scanner, or a Zeiss point scanning Confocal microscope LSM700.

Slices were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) overnight, then incubated with a blocking solution (2% milk in PB supplemented with 0.3–1% Triton X-100). Streptavidin-Cy3 or Cy5 conjugate (1:500; Life Technologies) was used for biocytin staining, and DAPI (1:1000) to stain nuclei. Sections were imaged using a pseudo-confocal Olympus IX81 microscope, and Volocity software for analysis, or a Zeiss LSM 710 confocal microscope. Retrobeads-labeled MEC-projecting neurons of the PrS were visualized in stacks of confocal images of NeuN-stained 60-µm sections and counted manually, in four ipsilateral and three contralateral sections from two mice. Their laminar distribution was quantified in each section (total 100% per section). Retrobeads labeled GABAergic neurons were counted in three sections from one GAD67-GFP mouse and from one SstCre::tdTomato mouse. The Neurolucida software was used for 3-D computer-aided morphologic reconstruction of biocytin-filled neurons as in (Simonnet et al., 2013 (link)).

HeLa cells were plated at a density of 1×10⁵ per well on fibronectin-coated glass coverslips in a six-well plate 6 h prior to transfection. Cells were transfected with 0.8 μg SKIP DNA (CB6 expression vectors) using Effectene transfection reagent (Qiagen). The cells were then incubated at 37°C for 16 h. Cells were fixed at −20°C with 100% methanol for 10 min. This was followed by blocking with 1% BSA in PBS for 20 min. Cells were washed three times with PBS. Cells were then probed with the appropriate antibody (LAMP1, Cell Signaling, D2D11, 1:200 or anti-Myc, Sigma, 9E10, 1:400) diluted in blocking solution (1% BSA in PBS). After 2 h at room temperature, cells were washed three times for 5 min each time with PBS followed by incubation with a secondary fluorescent-protein-conjugated antibody (in blocking solution) for 30 min. Coverslips were washed three more times with PBS, placed cell side down in Fluor save reagent (Calbiochem). Widefield fluorescence images were collected using a Zeiss Olympus IX-81 microscope with a 40× objective running Metamorph software. Confocal images were collected using a Nikon A1 system with a 100× objective running NIS Elements.