Samples were imaged using Leica confocal microscope (Leica TCS SP5X) with 63X oil immersion objective, with a Z-stack collected for each channel (step size 0.2 μm). Images were processed using ImageJ software (Version: 2.0.0-rc-65/1.52q, build: 961c5f1b7f) and individual nuclei were identified by DAPI staining. Nuclear lamina and nuclear speckles were identified by LaminB1 and SON immunostaining, respectively. 3D reconstructions of cells were conducted via the Imaris x64 (v 9.2.1) software (Bitplane AG, Switzerland). DNA FISH dots for probe signal were created at the location of the highest fluorescence intensity via the Spots Tool. Spot diameter ranged from 250 nm to 300 nm. Nuclear lamina surfaces were automatically detected from LaminB1 immunostaining with the Surfaces tool. Distance from the center of the FISH spot to the closest nuclear lamina surface was quantified using the Measurement Points tool. 40–60 nuclei were analyzed for each experimental condition. In the case that the generated DNA FISH spot was embedded in the nuclear laminar surface, distance to the lamina was quantified as zero.
Leica tcs sp5x
Manufactured by Leica camera
Sourced in Spain, United Kingdom
The Leica TCS SP5X is a high-performance confocal microscope system designed for advanced imaging applications. It features a flexible and modular design that allows for customization to meet the specific needs of researchers and laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Nb400 144, by Novus Biologicals (1 mentions)
Alexa fluor 568 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions)
Nucgreen dead 488 reagent, by Thermo Fisher Scientific (1 mentions)
β tubulin 3, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
4 protocols using leica tcs sp5x
1
3D Quantification of Chromosomal Positioning
2
Visualizing CD36 and N-cadherin in Liver Tissue
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Paraffin‐embedded liver biopsy sections (4 μm thick) were co‐incubated with an anti‐CD36 antibody (NB400‐144, Novus) and anti‐N‐cadherin antibody (BP1‐48309, Novus), diluted to 1:200 and 1:25 respectively, following with the appropriated conjugated secondary antibodies Alexa Fluor® 568 goat anti‐rabbit IgG (A11011, Life Technologies) or Alexa Fluor® 488 goat anti‐mouse IgG (A11029, Life Technologies). The immunofluorescence‐mounting medium used was Fluoromont G® (BioNova cientifica). Representative images were taken using a confocal microscope Leica TCS SP5 X (Leica, Barcelona, Spain).
3
Quantification of Neuronal Morphology
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Cells were seeded in 12‐well plates and fixed with 4% formaldehyde in PBS for 15 min at room temperature (RT) and blocked in 3% BSA in PBS for 60 min at RT, followed by incubation in primary antibody overnight at 4°C and secondary antibody for 2 hr at RT. The antibodies' sources, catalogue numbers, and dilutions are listed in Table S1 . The morphological characters were quantified by a confocal laser scan microscope (Leica TCS SP5 X; Leica). Tuj1+, MAP2+, and DAPI+ cells were counted in a blinded manner from 10 randomly selected images (×10 magnification) for each condition. The length of the neurites was measured as previously described (Tegenge, Roloff, & Bicker, 2011 ). Briefly, 20–30 Tuj1+ neurons were traced and analyzed blindly for the total length of the neurites using Image J with the Neuron J. The number of terminals was counted via tracing. In order to avoid any ambiguity, neurons with overlapping neurites were excluded. The data presented are the mean of three independent experiments, shown as the mean ± standard error of the mean (SEM). For neurites length and number of terminals analysis, 250 cells per experiment were scored.
4
Immunofluorescence Staining of Cells
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After washing the cells with warm PBS three times for 5 min, the cells were fixed with 3.7% paraformaldehyde (104005, Merck Millipore, Darmstadt, Germany). For permeabilization of the cells, we used 1% Triton X-100 (Merck Millipore, Burlington, MA, USA) for 10 min at room temperature, followed by blocking in 10% normal goat serum (NGS, Thermofisher Scientific, Bleiswijk, The Netherlands) in PBS for 15 min at RT. The cells were incubated with the primary antibody in blocking solution ( -tubulin III 1:200, (Sigma Aldrich)) overnight at 4 °C. After three times for 5 min PBS washing steps, the cells were exposed to secondary antibodies (anti-mouse IgG (H + L) Alexa 647 1:200, Thermofisher Scientific, Bleiswijk, The Netherlands) and 2 drops/mL NucGreen® Dead 488 reagent (R37109, Thermo Fisher Scientific)) for 3 h at room temperature. Images were obtained with a Leica TCS SP5X confocal laser scanning microscope (Leica TCS SP5X, Leica, Milton Keynes, UK). The images were built up by using proper excitation wavelengths for the different applications.
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