Nis elements interface

PC-3 cells (ECACC 90112714) were maintained in RPMI medium (GIBCO, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum and penicillin/streptomycin. For live-cell confocal imaging, 400,000 cells were seeded on 1.5 high-tolerance 25-mm glass coverslips (Warner Instruments) 24 h prior to transfection. Caveolin1–red fluorescent protein, Cavin1–green fluorescent protein (GFP), and Cavin1-ΔDR1-GFP were transiently transfected using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions 16 to 24 h before the experiment. Live-cell experiments were performed using a growth chamber (37 °C, 5% CO₂) connected to an Eclipse Ti-E inverted microscope (Nikon Instruments) equipped with a DU897 ANDOR electron multiplying charge-coupled device camera (Oxford Instruments), Nikon CFI Plan Apochromat 60× oil (numerical aperture [NA] 1.40) differential interference contrast objective, and Nikon CFI Plan Apochromat 100× (NA 1.49). The total internal reflection fluorescence objective was controlled by an NIS Elements interface (Nikon Instruments). Images were prepared using ImageJ (38 (link)) and Photoshop CS6 (Adobe).

Sections ranging from Bregma AP –0.1 mm to AP –0.7 mm for each animal (n = 84 animals; n = 211 sections) were selected for fluorescence-based microscopy imaging. Images were acquired using a TxRed filter (excitation = 540–580 nm; emission = 600–660 nm) on a Nikon Eclipse Ti-E inverted microscope with a DU897 ANDOR EMCCD camera controlled by Nikon NIS Elements interface, equipped with Nikon CFI Plan Apochromat ×20 (N.A 0.75) objective. Prior to analysis, all the images were aligned using Amira-Avizo Software (version 6.3.0, Thermo Fisher Scientific). A region of interest (ROI) was selected based upon a significant VBM cluster in SSp-ul described in this study. The ROIs were manually positioned and saved for each section using FIJI (Schindelin et al., 2012 (link)). Signal-to-noise (specific myelin immunoreactivity versus background fluorescence) was determined by the segmentation of each image using FIJI’s Multi Otsu Threshold plugin using three different levels of classification. This plugin is based on Otsu’s original method but also implements an algorithm described by Liao and Chung (Liao et al., 2001 (link)). The specific signal was quantified for each section per individual to calculate a mean immunoreactive value for each subject (n = 12 per time point).