Dfc345 fx

IGR-NB8 cells were seeded on coverslips coated with poly-l-lysine (Sigma-Aldrich) in 24-well cell culture plates and treated with CUDC-907 for 48 h. NET and DAT proteins were revealed after incubation with primary antibodies (ref. NET17-1 and ab1766) and Alexa FluorTM-coupled secondary antibodies (ref. A11001 and A 11008, anti-mouse and anti-rabbit respectively) from ThermoFischer. Specificity was assessed by primary antibody omission. The immunofluorescence protocol is described elsewhere [22 (link)]. Cells were imaged using a fluorescence microscope (Leica DFC 345 FX) and then analyzed with LAS AF Lite software (Version 2.6.0, build 7266). Signal was quantified using the ImageJ software.

For analysis of GFP, mCherry or SNB-1::GFP, live animals were anesthetized using 1% (v/v) 1-phenoxy-2-propanol in M9 buffer and visualized using a 40× magnification oil-immersion lens and an epifluorescent microscope (Leica CFR5000). A CCD camera (Leica DFC345 FX) was used for documentation. Images were analyzed and scale bars applied using Leica Application Suite for Advanced Fluorescence (LAS-AF) software. Termination defects were quantified by manually scoring juIs76 (P_unc-25::GFP) for dorsal cord tips, and bggIs6 (P_unc-25::mCherry) for DD5. Each genotype was quantitated by manually scoring three counts of 10 to 20 animals from two or more independent experiments.

Digital photomicrographs were captured under bright-field (DIC) or fluorescent light (fluorescence IHC) using a Leica DFC345FX camera attached to a Leica DMI8 microscope using Leica Application Suite X (LASX) imaging software (ver. 2.0.0.14332.2). All analyses were performed on a minimum of three eyes per genotype and a minimum of three photomicrographs per eye. Statistical significance was calculated using unpaired two-tailed Student's t-tests (to compare two samples) or one-way ANOVA (for multiple sample comparisons, with a Tukey post hoc analysis) using GraphPad Prism Software version 5.0 (GraphPad Software). Error bars represent standard error of the mean (s.e.m.).

For analysis of RPM-1::GFP, live adult animals were anesthetized using Levamisole in M9 buffer and visualized using an epifluorescent microscope (Leica CFR5000) with a 40× magnification oil-immersion lens. Images were acquired using a CCD camera (Leica DFC345 FX). Ten animals per strain were observed across two independent days with representative images shown in Figure 5.

Chromosome preparations were analyzed under a Leica DM 4000B microscope with a 100x objective. Images were taken with a Leica DFC 345 FX camera using Leica Application Suite 3.7 software with an Image Overlay module.

Neutral comet assays were performed using CometAssay (Trevigen) according to the manufacturer's protocol with minor modifications (Carvajal-Maldonado et al., 2019 (link)). Upon harvesting, cells were suspended at 3×10⁵ cells/ml in cold PBS before combining with LMAgarose (Trevigen, 4250-50-050-02), spread onto a comet slide (Trevigen, 4250-200-03) and allowed to dry. Slides were placed in lysis solution (Trevigen, 4250-050-01) at 4°C for 1 h. Lysed slides were immersed in 1× TBE buffer (0.1 M Tris base, 0.1 M boric acid, 2.5 mM EDTA) for 30 min before electrophoresis at 25 V for 30 min at 4°C. Slides were washed in DNA precipitate solution (1 M ammonium acetate, 95% EtOH) for 30 min, followed by a fixing step in 70% ethanol for 30 min, and dried overnight at RT. Comets were stained with 1× SYBR Gold (Thermo Fisher Scientific) for 30 min. Images were acquired with a fluorescence microscope (Leica DMU 4000B; 63×/1.40-0.60 NA oil) with a Leica DFC345FX camera. At least 150 comets were scored in each experiment using the OpenComet plugin in the ImageJ analysis software.

Immunofluorescence was evaluated in three representative alternate sections of the lumbar spinal cord per specimen. After blocking with 3% BSA (bovine serum albumin) in 0.1 M PB for 45 min, slides were incubated with primary antibodies against GFAP, Iba1, synaptophysin, MHC-I, GAD65, VGLUT-1, and AMPA receptor (Table 3) diluted in an incubation solution (1.5% BSA and 0.2% Tween in 0.01 M PB) and incubated for 4 h at room temperature. After washing with 0.01 M PB, the secondary antibodies (Table 3) were applied and incubated for 45 min. Sections were then rinsed in 0.01 M PB and mounted in a mixture of glycerol/PB (3:1).
For quantification, one image of each side (right and left) was acquired for each slide, totaling 3 per animal, using a Leica fluorescence microscope (DM 5500, Wetzlar, Germany) equipped with a digital camera (DFC 345 FX, Wetzlar, Germany) using specific filters according to the secondary antibodies. A quantitative evaluation of labeling was carried out using the integrated density of pixel measurements in a fixed area corresponding to the ventral horn (Figure 1), as described by Oliveira et al. (2004) (link).
Quantification was performed with ImageJ software (version 1.33u, National Institutes of Health, USA). The integrated density of pixels was calculated for each image, and the mean value for each experimental animal was calculated.