Nis elements advanced research software

After the 48-h co-culture experiment, the cells were washed three times with ice-cold Dulbecco’s phosphate-buffered saline solution (DPBS, Gibco, Carlsbad, CA, USA). Cells were subsequentially incubated with 5 µg/mL Hoechst 33342 (H3570, Thermo Fisher Scientific, Waltham, MA, USA) for 15 min before being imaged using a Nikon Eclipse Ti2 microscope (Nikon Instruments, Tokyo, Japan) equipped with a Yokogawa CSU-W1 Confocal Scanner Unit (Yokogawa, Tokyo, Japan) and Hamamatsu C13440-20CU ORCA Flash 4.0 V3 Digital CMOS camera (Hamamatsu photonics, Hamamatsu, Japan). Microscope operation and image acquisition were performed using Nikon NIS-Elements Advanced Research software (Nikon Instruments, Tokyo, Japan). ImageJ2 Version 2.3.0/1.53q (NIH, Bethesda, MD, USA) was used to process the obtained images.

At the end of the behavioral analysis, mice were perfused transcardially with ice-cold PBS with 5 U/mL sodium heparin (Hospira). Brains were post-fixed in 4% PFA for 48 h, cryo-protected in sucrose, and 8 µm coronal sections mounted on SuperFrost slides (Fisher Scientific; 12–550-15), were post-fixed in 4% PFA for 1 h, blocked in 2% bovine serum albumin, 10% normal goat serum and 0.1% saponin in PBS and incubated with rabbit anti-synaptophysin (1:1000, Millipore; AB9272) or rabbit anti-PSD95 (1:1000, Abcam; AB18258), followed by Alexa 488 goat anti-rabbit (1:500, Invitrogen; A-11043) or Alexa 647 goat anti rabbit (1:500, Thermo Fisher Scientific; A-21245). As a negative control, the primary antibody was omitted. Fluorescence was visualized using the Nikon A1R Confocal Microscope (Nikon Instruments Inc., Melville, NY, USA) using 40X objective. The average number of synaptophysin and PSD95 positive puncta were quantified in the CA1 of the hippocampus in three regions of interest using the spot detection feature of the Nikon NIS-Elements Advanced Research Software (Nikon Instruments Inc., Melville, NY, USA).

HeLa cells expressing red fluorescent protein-tubulin (RFP-tubulin) and GFP-histone H2B (15 (link)) were silenced with siRNAs using Lipofectamine RNAiMAx (Life Technologies). 48 h after transfection, cells were placed in complete DMEM without phenol red in a temperature- and CO₂-controlled incubation chamber (Solent Scientific Ltd), and images were acquired every 10 min using a Nikon Ti Eclipse inverted microscope, a Nikon ×10 Plan Fluor 0.3 numerical aperture Ph1 lens, and Nikon fluorescence filter sets for GFP and RFP (Nikon UK Ltd). Image acquisition was performed using a Photometrics Coolsnap HQ2 charge-coupled device camera (Photometrics Ltd) and Nikon Nis-Elements advanced research software (Nikon Instruments Europe). A combination of brightfield, RFP-tubulin, and GFP-H2B images was used to determine cells entering mitosis, progressing, exiting mitosis, and undergoing cell death. Using these criteria, we were able to determine the total number of cells attempting mitosis over a 10-h time course separated into 10-min time frames. Cells termed “successfully completing mitosis” entered, progressed, and exited mitosis without exhibiting any defects. Cells initiating mitosis and either exhibiting cell death within or shortly after mitosis or failing to complete cytokinesis were deemed to be cells attempting but failing to successfully complete mitosis.

Brain sections (35 µm) were maintained at −20 °C in cryoprotectant, stained while free-floating, and mounted to glass slides for imaging, using a “primary antibody delete” (secondary antibody only) stained section to establish background fluorescence limits. Fluorescent immunohistochemical images were collected using an Olympus BX61 confocal microscope and Fluoview 1000 software (Melville, NY). Quantitative fluorescence measurements were thoroughly monitored using standard operating imaging parameters to ensure that images contained no saturated pixels. For quantitative comparisons, all imaging parameters (e.g., laser power, exposure, and pinhole) were held constant across specimens. Confocal images were analyzed using Nikon NIS-Elements Advanced Research software (Version 4.5, Nikon, Melville, NY). A minimum of six images per tissue slice were analyzed per animal, averaging 9–15 neurons per 60–100× image (approximately 180 cells per animal, per histological stain). 20× magnification was used to generate montage imaging of the ventral midbrain, for which the entire SN was analyzed per image using anatomical region of interest (ROI) boundaries. Results are reported as a measure of puncta within TH-positive cells, either number of objects (# of objects), or area per neuron in square pixels (px²), generated by Nikon Elements Advanced Research software.

Stereological analysis of dopamine neuron number in the SN was achieved using an adapted protocol from Tapias et al.^{45 (link)} and Tapias and Greenamyre^{46 (link)} as reported in De Miranda et al.^{20 ,47 (link)} employing an unbiased, automated system as an alternative to the optical fractinator method. Briefly, 35 μm coronal nigral tissue sections (1/6 sampling fraction encompassing the volume of the entire SN) were stained for TH and counterstained with DAPI and NeuroTrace Dye (640; Life Technologies) and imaged using a Nikon 90i upright fluorescence microscope equipped with high N.A. plan fluor/apochromat objectives, Renishaw linear encoded microscope stage (Prior Electronics) and Q-imaging Retiga cooled CCD camera (Center for Biological Imaging, University of Pittsburgh). Images were processed using Nikon NIS-Elements Advanced Research software (Version 4.5, Nikon, Melville, NY), and quantitative analysis was performed on fluorescent images colocalizing DAPI, TH, and Nissl-positive stains. Results are reported as the number of TH-positive cell bodies (whole neurons) within the SN.