All images were obtained using a Zeiss LSM 510 confocal or a Leica SP5 confocal except for Fig. 6D and Supplement to Fig. 5G , which was obtained using a Leica DMI6000 epi-fluorescence system with de-convolution (ImageQuant). All images were cropped, rotated and processed using Adobe Photoshop. For brightness/contrast the Auto Contrast function was used. All brightness/contrast adjustments were applied equally on the entire image. All x/z optical sections were obtained on a Leica SP5 confocal. Images for x/z sections are maximum projections of 1–3 y-sections of 0.5 micron intervals or less. All image quantifications were performed using Image J.
Sp5 confocal
Manufactured by Leica camera
Sourced in Germany
The Leica SP5 confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features a modular design that allows for customization to suit diverse research needs. The SP5 confocal provides accurate and reliable data acquisition, with the ability to capture high-resolution images and perform advanced imaging techniques.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Eos t3, by Canon (2 mentions)
Axioskope, by Zeiss (2 mentions)
Axiocam camera, by Zeiss (2 mentions)
Micrometer, by Avantor (2 mentions)
Na 1.3 glycerol objective, by Leica camera (2 mentions)
Las af software, by Leica camera (2 mentions)
Anti dsred, by Takara Bio (2 mentions)
Axio imager, by Zeiss (2 mentions)
Sheep anti gfp, by Bio-Rad (1 mentions)
Dmra2 widefield epifluorescence microscope, by Leica camera (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Deltavision system, by GE Healthcare (1 mentions)
Ab150079, by Thermo Fisher Scientific (1 mentions)
D1306, by Thermo Fisher Scientific (1 mentions)
Ma5 12960, by Thermo Fisher Scientific (1 mentions)
Ab150077, by Abcam (1 mentions)
Ab150116, by Abcam (1 mentions)
Matlab, by MathWorks (1 mentions)
Dmra2 upright microscope, by Leica camera (1 mentions)
39 protocols using sp5 confocal
1
Confocal Microscopy Imaging Protocol
2
Quantitative Analysis of Muscle Histology
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Images of hematoxylin and eosin stained muscle sections were captured from a Nikon 800 microscope with 10× or 20× Plan Apo objectives and Canon EOS T3 camera using EOS Utility image acquisition software. Fluorescent images of muscle sections and single myofibers were wither captured using Leica SP5 confocal equipped with 40×/1.25 Plan Apo oil objectives using Leica image acquisition software, or a Zeiss Axioskope equipped with a 40×/0.5 Plan Apo oil objective and Axiocam camera using Zeiss image acquisition software. Identical exposure times were used and images were processed and scored with blinding using ImageJ64. If necessary, brightness and contrast were adjusted for an entire experimental image set. For quantification of polarized cell markers (Par3, m-cadherin, pp38), all authors individually scored each set of images with blinding using ImageJ64, only those that were agreed upon by all were included. For the rest, Imaris (Bitplane) was used for three-dimensional rendering of fluorescence data for quantification of polarization. A subset was also randomly selected for quantitative analysis via Imaris to confirm authors’ scoring. Cell number, fiber diameter, fiber number, and fiber cross sectional area were determined using ImageJ64 or Fiji using images of a micrometer (VWR) taken under the same magnifications as the sample images as references for imaging field sizes.
3
Immunofluorescent Labeling of Adult Brains
4
Confocal Imaging of Alexa647 Samples
5
Quantifying Ploidy in Drosophila Wing Discs
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For quantification of ploidy in Figure 3 , wing imaginal discs from Tb+ 3rd instar larvae were dissected and fixed as described (Schwed et al. 2002 (link)). Discs were labeled with rabbit anti-dsRed (Clontech, 632496) (1:400) and secondary anti-rabbit Alexa Fluor 568 (1:500) (Invitrogen), and stained with DAPI (0.5μg/ml). Discs were imaged on a Leica SP5 confocal and Leica DMRA2 widefield epifluorescence microscope. ImageJ was used to quantify nuclear area and total DAPI fluorescence. The nuclear area and DAPI intensity of cells within the RFP+ dpp expressing stripe were normalized to cells outside of the stripe in the same wing discs (RFP + cells / RFP- cells in Figure 3 ). The cells in the wing pouch area of each wing disc were scored, excluding the zone of non-proliferating cells, which are arrested in G1 and G2 phases of the cell cycle (Johnston and Edgar 1998 (link)).
6
Neuronal Actin Dynamics Visualization
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Neuronal GFP-Actin expression was obtained by electroporation before plating, employing a BioRad Cell electroporator system. Approximately 4x106 cells and 10 μg of plasmid were mixed in BioRad electroporation buffer (BioRad). An exponential discharge protocol (220 V, 950 μF, resistance fixed to infinitum) was employed. Spines were visualized by transfection with a plasmid encoding the GFP protein fused to chick β-actin under the control of the platelet-derived growth factor promoter region (kindly provided by Y. Goda, MRC Cell Biology Unit, University College London, London, UK) [45 (link)]. Transfected cultures were fixed with 4% paraformaldehyde-PBS, washed and mounted in Mowiol. Images were obtained in a Leica SP5 Confocal, acquired in stacks (pixel size 60 nm with 0.1 μm Z step) and processed as above. Spine density represents the number of spines per 100 μm of dendritic length.
7
Confocal Imaging of Zebrafish Hindbrain
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Embryos were mounted in low melting point agarose and confocal time-lapse movies were made at 28.5°C as previously described using a Leica SP5 confocal and water dipping x25 and x40 objectives (Tawk et al., 2007 (link)). Data was collected from the hindbrain regions. Some adjacent cells have been cropped from the images to increase clarity of the cells of interest. Images were processed using Volocity and ImageJ.
8
Visualizing Cellular Response to Stress
9
Multicolor Immunostaining of Cardiac Microtissues
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Microtissues were washed 3× times with PBS and then fixed overnight in 10% formalin at 4 °C. Following 3× washes with PBS, fixed microtissues were incubated for 4 h in permeabilization solution containing 0.2% Triton X-100 in PBS supplemented with 2% BSA, 320 mM sucrose and 6 mM magnesium chloride. Before immunostaining, microtissues were blocked in PBS containing 2% BSA for 2 h. Next, microtissues were stained with primary antibodies for cardiac troponin-t (cTnT) (Thermofisher MA5-12960, 1:200) and vimentin (Thermofisher MA5-16409; 1:200) to visualize cardiomyocytes and fibroblasts, respectively. To visualize sarcomeres, samples were stained with anti-sarcomeric alpha actinin antibody (abcam 137346; 1:200). To visualize gap junctions, samples were stained with connexin-43 (Thermofisher 71-0700; 1:200). Primary antibodies were diluted in PBS containing 3% BSA and hydrogels were stained at 4 °C for 72 h. Next, the secondary antibodies Alexa Fluor-488/594/647 IgG H&L (1:200; Abcam ab150077, ab150116, ab150079) were added at 4 °C for 72 h. Finally, samples were washed 3× in PBS followed by DAPI staining (1:2000; Invitrogen D1306, in PBS) for 30 min at room temperature. A Leica SP5 confocal was used to acquire z-stack images.
10
Quantifying Tumor Spheroid Nuclei
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Images were taken using SP5 Confocal (Leica). Single projection images of 4–6 z-stack sections from at least 15–20 tumorspheres were constructed and analyzed in the Matlab software package (version R2016b by Mathworks, Natick, MA). Individual cell nuclei were segmented using regional intensity maxima and watershed thresholding on the DAPI channel, and then scored by the presence of relevant IF signal above a global intensity threshold in the area immediately surrounding each nucleus. For BrdU images, signal was calculated in each nucleus around intensity maxima. Quality control images were also created by superimposing the cell scoring mask on the raw image data.
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