Fluoview fv1000 confocal laser scanning biological microscope

For phase contrast imaging, testes from a minimum of five males per strain were dissected in Hoyle’s medium (Baccetti et al., 1979 (link)), mounted in 1X PBS, and viewed using an EVOS Fl Auto Imaging System. For confocal microscopy, testes were dissected, fixed with 4% formaldehyde in 1X PBS, and stained with rhodamine phalloidin (Cytoskeleton, Inc.) at 1:1000 in 1X PBS (Bonaccorsi et al. 2011 (link)). At least thirteen males per strain were dissected, typically both testes were examined. The testes were placed in depression slides with approximately 20µl of 2µg/ml DAPI in 1X PBS. Samples were viewed using an Olympus FV 1000 fluoview confocal laser scanning biological microscope.

Tissue section images were obtained at 20× magnification using an FV1000 Fluoview Confocal Laser Scanning Biological Microscope (Olympus, Dublin, Ireland). All acquisition settings remained identical across all sections allowing intensity quantification and comparison. Images were processed using ImageJ (W. RasBand, National Institute of Health, Bethesda, USA). Three to six sections were imaged and analyzed per animal, resulting in 18 to 36 sections per control and experimental groups. For in vivo quantification with respect to the distance from the electrode insertion site, ROI boxes with a constant width of 200 µm and length of 50 µm were used every 50 µm and up to 250 µm radially outward from the probe void. The mean intensity and percentage of the stained area was measured in each ROI box for GFAP and Iba1 stained sections and NeuN⁺ nuclei were counted using the “analyze particles” ImageJ tool. For each NeuN stained image, the distance of the 6 nearest NeuN⁺ nuclei from the electrode void was measured using the draw line tool. Finally, data were averaged for each group and plotted by mean ± SEM as a function of distance from the electrode site. The researcher performing the quantitative tissue analysis was blind to the different group identities.

Z‐stack images were obtained at a 60× magnification using an FV1000 Fluoview Confocal Laser Scanning Biological Microscope (Olympus, Dublin, Ireland). All acquisition settings were held constant, allowing intensity quantification and comparison. A minimum of five fields of view (FOV) was acquired per technical replicate, resulting in ≈30 images per control and experimental groups. Images were processed using ImageJ (W. RasBand, National Institute of Health, Bethesda, USA). The stacks obtained from each FOV were projected into a single image using the maximal intensity projection, then the individual channels were isolated and quantified separately. Intensity, area and number of positive cells were automatically counted after thresholding the image as previously described by Healy et al.^[157] For each specific stain, the best automatic thresholding methods were assessed using the “Threshold Check” plugin. Specifically, the following methods were used to convert the image to binary: renvy and yen for astrocytes, moments, and maxentropy for microglia, li, and mean for neurons, minimum for DAPI. Obtained values were normalized either by the staining area or according to the total cell number in the FOV, automatically quantified with the DAPI signal.