After DNA transfection, MCF10A cells or HeLa cells were seeded to fibronectin coated coverslips (10 μg/cm2) for 2 h spreading at 37 °C. Adherent cells were fixed with 4% formaldehyde and stained with anti-vinculin antibody (Sigma) or anti-GFP antibody (abcam) and anti-HA antibody (Cell Signaling) or anti-Myc antibody (Cell Signaling) and anti-ILK antibody (abcam) followed by goat anti-mouse antibody Alexa-488-conjugated (abcam) or goat anti-chicken antibody Alexa-488-conjugated (abcam), goat anti-rabbit Alexa 568-conjugated (abcam) and Alexa Fluor 647 phalloidin (Invitrogen). Coverslips were then mounted with Prolong Diamond Antifade Reagent with DAPI (Fisher) overnight and visualized with Leica TCS-SP5 II upright confocal microscope (Leica Microsystems, GmbH). Images were followed with ImagePro 10 software processing for spreading area and FA size/number quantification.
Tcs sp5 2 upright confocal microscope
Manufactured by Leica
Sourced in Germany
The Leica TCS SP5-II upright confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features a modular and flexible design, allowing for customization to meet the specific needs of various research and analysis tasks. The system provides high-resolution, optical sectioning capabilities, enabling detailed examination of biological samples and materials at the cellular and subcellular levels.
Automatically generated - may contain errors
Lab products found in correlation
Anti gfp antibody, by Abcam (2 mentions)
Anti vinculin antibody, by Merck Group (2 mentions)
Anti ilk antibody, by Abcam (2 mentions)
Anti myc antibody, by Cell Signaling Technology (1 mentions)
Anti ha antibody, by Cell Signaling Technology (1 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (1 mentions)
Alexa 647 phalloidin, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Tcs nt confocal laser scanning microscope, by Leica (1 mentions)
Superdex 75 10 300 gl, by Cytiva (1 mentions)
Alexa 488 phalloidin, by Merck Group (1 mentions)
Mirax desk scanner, by Zeiss (1 mentions)
Hcx pl apo, by Leica (1 mentions)
Lsm 5 live microscope, by Zeiss (1 mentions)
Metamorph, by Molecular Devices (1 mentions)
80i microscope, by Nikon (1 mentions)
Asp6025 processor, by Leica (1 mentions)
7 protocols using tcs sp5 2 upright confocal microscope
1
Immunostaining of Focal Adhesions in Transfected Cells
2
Visualizing integrin-mediated adhesion complexes
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HEL cells transfected with EGFP-tagged constructs were stimulated with 800 nM PMA for 5 min and incubated with immobilized fibrinogen in duplicate, for 1 h or 2 h at 37 °C in serum-free medium. After extensive washing with PBS, the adherent cells were fixed with 4% PFA and stained antivinculin antibody (hVIN-1 clone, Sigma-Aldrich, St. Louis, MO), followed by secondary antimouse goat F(ab) fragment antibody Alexa 568-conjugated (Thermo Fisher Scientific) and with Alexa 647-phalloidin (Thermo Fisher Scientific). The cells were visualized with 40x and 63X objectives using a Leica TCS-NT laser scanning confocal microscope and the cell area was measured for 150 cells with ImageJ software.
HeLa cells after CRISPR or cell sorting were seeded to fibronectin-coated coverslips (10 µg/cm2) for 2 h spreading at 37 °C. Adherent cells were fixed with 4% PFA and stained with anti-vinculin antibody (Sigma) or anti-GFP antibody (Abcam) and anti-ILK antibody (abcam) followed by goat anti-mouse antibody Alexa 488-conjugated (abcam) or goat anti-chicken antibody Alexa 488-conjugated (Abcam) and goat anti-rabbit Alexa 568-conjugated (abcam). Coverslips were then mounted with Prolong Gold Antifade Reagent with DAPI (Fisher) overnight and visualized with Leica TCS-SP5 II upright confocal microscope (Leica Microsystems, GmbH, Wetzlar, Germany).
HeLa cells after CRISPR or cell sorting were seeded to fibronectin-coated coverslips (10 µg/cm2) for 2 h spreading at 37 °C. Adherent cells were fixed with 4% PFA and stained with anti-vinculin antibody (Sigma) or anti-GFP antibody (Abcam) and anti-ILK antibody (abcam) followed by goat anti-mouse antibody Alexa 488-conjugated (abcam) or goat anti-chicken antibody Alexa 488-conjugated (Abcam) and goat anti-rabbit Alexa 568-conjugated (abcam). Coverslips were then mounted with Prolong Gold Antifade Reagent with DAPI (Fisher) overnight and visualized with Leica TCS-SP5 II upright confocal microscope (Leica Microsystems, GmbH, Wetzlar, Germany).
3
Actin Polymerization and Visualization
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Rabbit skeletal muscle lyophilized actin (Cytoskeleton, Inc) was dissolved in G-buffer (5 mM Tris-HCl, pH 8.0), 0.2 mM CaCl2, 0.2 mM ATP, 0.5 mM DTT, 1 mM NaN3) to the concentration of ~8 μM, incubated on ice for 1 h and injected into Superdex 75 10/300 GL (Amersham Biosciences) gel filtration column. Monomer fractions were collected and used for the experiment. Usually, the fractions contained G-actin concentration of 3–5 μM. Then we polymerized 1 μM G-actin by addition of salts (50 mM KCl, 2 mM MgCl2, 1 mM ATP) in the presence of IPP buffer or IPP protein (1:1 molar ratio) and the filaments were labeled with ~1 μM Alexa 488-phalloidin (Sigma). Prior to the visualization, 1 μl of sample was added to 9 μL of F-buffer (G-buffer above plus salts) and applied to a cover slip coated with poly-L-lysine (0.01%). Widefield images were acquired using a Leica DM5000B upright fluorescence microscope (Leica Microsystems, GmbH, Wetzlar, Germany), equipped with a Retiga SRV camera and QCapture Pro software (QImaging, Surrey, BC Canada). Confocal images were acquired using a Leica TCS-SP5 II upright confocal microscope (Leica Microsystems, GmbH, Wetzlar, Germany).
4
Quantitative Confocal Microscopy of Gut Microbiome
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Images were acquired with a Leica TCS SP5-II upright confocal microscope using a 63x oil immersion lens (NA 1.4, HCX PL APO) with or without digital zoom as a series of short Z-stacks. Maximum intensity projection processing of Z-stacks was done in Fiji (ImageJ) software. Mucus layer thickness was measured using the Leica distance measurement tool (LASAF). The width of the inner mucus layer was determined by the average of 4 measurements per field with 4 fields measured per section. Whole tissue images were digitally scanned using the Zeiss Mirax Desk Scanner with 20x/0.8NA objective. Bacterial distance analysis was performed on colon images taken at 63x magnification with a 4x digital zoom by determining the XY coordinates of each bacterial cell in MetaMorph (Molecular Devices) software and measuring the distance from their center. For quantification of bacterial density and invasion into the mucus layer, whole tissue cross-sections were tile scanned in short Z-stacks using an inverted laser scanning confocal Zeiss LSM 5-Live microscope at 63x magnification. For bacterial quantification, a threshold based on the RGB color combination and intensity of each bacterial species was generated with the color thresholding option in MetaMorph. Thresholded objects of 1μm in size were counted as a single bacterial cell with MetaMorph’s integrated morphometric analysis tool.
5
Immunostaining of Mouse Brain and 3D Spheroids
6
Multicolor Fluorescent In Situ Hybridization
7
Actin and Nuclei Visualization in 48 hpf Embryos
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48 hpf embryos were fixed with 4% paraformaldehyde overnight at 4℃. Whole-mount embryos were embedded in 4% agarose and cut in 150 μm thick sections with Leica VT1OOOS Vibratome. Sections were washed 3 times for 15 min with PBT (0.8% Triton X-100 in PBS) and then incubated for 3 h at room temperature in phalloidin-633 (1:300 in PBT) for filamentous actin staining and 20 min at room temperature for DAPI-stained nuclei. Imaging was performed using Leica TCS SP5II upright confocal microscope. Images were capture with LAS AF Software, analyzed in ImageJ Software and presented as maximum projection of a set of z-stacks for each stained tissue section.
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