Plan apochromat 20x na 0

Sagittal sections of lumbar spinal cord were immunostained as described above and z stack images were taken on a Zeiss LSM 700 confocal microscope using a 20X lens (Plan-Apochromat 20X/NA 0.8). Analysis was limited to the LTMR-RZ, using IB4 (IIiv border) as an upper limit and 250μm below IB4 as a lower limit. Confocal image stacks were loaded into the Neurolucida 360 software. Specific neurons from each image stack were reconstructed using the user-guided reconstruction tool. Reconstructions were saved and opened in Neurolucida Explorer software for morphological analysis. Basic information detailing somatic and dendritic measurements were retrieved from the reconstructions using Neurolucida software and graphed with GraphPad Prism. Sholl-based metrics detailed in Figure S3 including: Enclosing radius, Sum of Intersections, Critical Value (Nm), Critical Radius (Rc), Mean Value (Nav), Centroid Value, Centroid Radius, Ramification Index (RI), Regression Coefficient (k), Branching Index (BI), were obtained by analyzing intersection-based sholl data obtained in Neurolucida with MATLAB script written using previously described formulas (Ferreira et al., 2014 (link), Garcia-Segura and Perez-Marquez, 2014 (link), Rajković et al., 2016 (link)). The depth location within the LTMR-RZ was measured from the bottom of the IB4⁺ lamina IIiv using ImageJ software.

Sagittal sections of lumbar spinal cord (50 μm) were immunostained as described above and z stack images were taken on a Zeiss LSM 700 confocal microscope using a 20X lens (Plan-Apochromat 20X/NA 0.8). Low-level expression of synaptophysin-tdTomato in cellular cytosol was used to locate sparsely labeled cells and follow neurites to all tdTom⁺ synapses. ImageJ software and multipoint tool was used for marking synapses and exporting coordinates; center of cell soma and lamina IIiv border (using IB4 binding) were also marked and measured. Synaptic coordinates were then converted into their location in the dorsal-ventral axis relative to IB4. Cells with somas residing outside of the LTMR-RZ were not included in the analysis. A minimum total of 10 cells from 3 animals was used in this synaptic distribution analysis.

Equal amount of HEK293T cells stably expressing DSP1-7 or DSP8-11 were mixed (1.25 × 10⁵ each) and seeded on glass-bottom plates (12-well black, glass-bottom #1.5 H; Cellvis) pre-treated with 20 μg/ml of Poly-L-lysine. 24 hr later, the cells were transfected with 1 µg pCI::H2B-RFP or pCI::JUNO::H2B-RFP. 18 h after transfection, 4 × 10⁶ capacitated wild-type sperm cells in mHTF were added to the HEK293T cells and co-incubated for 4 hr after which they were washed with PBS, fixed with 4% PFA in PBS and stained with 1 µg/ml DAPI. In addition, a time course experiment was conducted where the cells were fixed at different time points. Micrographs were obtained as above, using wide-field illumination using an ELYRA system S.1 microscope (Plan-Apochromat 20x NA 0.8; Zeiss). The number of GFP-positive cells per 1000 nuclei was determined. Between 1000–2000 red nuclei were counted in each independent repetition (experimental point).