Nis elements basic research software

HEK cells were transfected with plasmids (either 50 ng or adjusted doses as stated in each figure), seeded on glass coverslips, and serum-starved overnight. Cells were fixed in 4% paraformaldehyde for 10 min and were washed with PBS several times. Cells were blocked with 5% goat serum in PBS for 1 h and incubated with mouse anti-HA antibody (1:1000) in 1% BSA in PBS for 2 h at room temperature. After several washing steps with PBS, HA antibody-bound receptors were labeled with Alexa Fluor 594–conjugated goat anti-mouse antibody (1:500) in 1% BSA in PBS for 1 h. Cells were mounted in ProLong Diamond Antifade Mountant with DAPI (ThermoFisher Scientific #P36971) for nuclear counterstaining. Fluorescence microscopy was conducted by 40 × oil objective (1.4 NA) on a Nikon Ti-E microscope equipped with a 16.2 MegaPixels DS-Ri2 camera and images were analyzed with Nikon NIS-Elements Basic Research software.

Testicular tissues were fixed in Bouin’s solution and embedded in paraffin for sectioning. In brief, the tissue slides were deparaffinized in xylene for 20 mins and then rehydrated with gradient ethanol (100%, 100%, 90%, 80%, 70%, and 50%), followed by staining with hematoxylin for 5 min and eosin for 1 min. After sequential dehydration with 50%, 70%, 80%, 90%, and 100% ethanol and transparency in xylene for 5 min, the tissue sections were sealed with neutral resin. The images were captured via a Nikon ECLIPSE 80i microscope (Nikon Instruments, Tokyo, Japan) equipped with a DR-Ri1 camera and processed with NIS-elements Basic Research software (Nikon Instruments).

Both the endpoint observation for conventional FISH and the real-time observation for μFISH were performed using an inverted microscope at 10×, 40× and 60× magnification (Nikon Eclipse Ti-E with objectives CFI Plan Fluor DLL 10×, ELWD 40× and ELWD 60×, respectively). An LED lamp (Sola, Lumencor) was used for illumination. Image acquisition was performed using an ORCA-flash 4.0 camera. For imaging, the NIS Elements Basic Research software (Nikon Instruments Europe, V.4.0) was used. Brightness and contrast adjustments of raw images as well as merging were done using the open-source FIJI (ImageJ) software (http://fiji.sc/Fiji), Supplemental Fig. S4.

Cells were differentiated in 96-well plates and then fixed at time-points with 5% formaldehyde (ThermoFisher) for 15 min, permeabilized with 0.5% Triton-X 100 (ThermoFisher) for 15 min, and blocked with 5% w/v BSA in PBS for 1 h. Primary antibodies include anti-MyoD G-1 (1:250, Santa Cruz Biotechnology, USA), anti-p21 F-5 (1:50, Santa Cruz Biotechnology), anti-p53 DO-1 (1:50, Santa Cruz Biotechnology), anti-phospho-p53 Ser 315 (1:50, Santa Cruz Biotechnology), anti-myogenin F5D (1:250, ThermoFisher), and anti-MHC MF20 (1:50, R&D Systems, USA). All primary antibodies were diluted in 5% w/v BSA in PBS and applied to blocked myotubes for 1 h. All primary antibodies were visualized using 1:200 Alexa Fluor 488 goat anti-mouse IgG (ThermoFisher). Nuclei were stained using NucBlue Live Cell Stain (ThermoFisher) for 15 min, and samples were preserved using Prolong Gold Antifade (ThermoFisher). All experiments were performed in triplicate. Images were acquired using an Eclipse Ts2R inverted fluorescence microscope (Nikon, Melville, NY, USA), a Zyla sCMOS camera (Andor, Belfast, UK), and NIS Elements basic research software (Nikon).

Cells were seeded at 1.5×10⁵ cells in a 60 mm dish (Fisher) and transfected 16 or 24 hr post seeding. Live cell imaging of EGFP-expressing cells was conducted with phase contrast and fluorescence microscopy using a Nikon ECLIPSE Ti inverted fluorescence microscope. Cells were observed over a five day period. Cells were scored either day 3 or 5 post-treatment for “neurites,” defined as neurite-like outgrowths ≧20 µm in length. Cells were scored at least by day 3, when the outgrowths were clearly visible. Neurite outgrowth length was measured with NIS-Elements Basic Research software (version 3.10, Nikon). At least three replicate transfections were performed and at least 100 cells were scored per replicate. Samples were scored blind with regard to treatment and were scored independently by at least two different individuals. Cells were scored into two categories: no neurites and neurites, except for the initial experiments where cells were categorized as <20, 20–40, 40–60, 60–80, 80–100, 100+ µm. Since this experiment showed neurite extensions longer than 20 µm to be characteristic of differentiation, subsequent studies focused on cells producing extensions >20 µm.