Tcs sp5 multiphoton laser scanning confocal microscope

Manufactured by Leica

Sourced in Japan, Germany

The Leica TCS SP5 is a multiphoton laser scanning confocal microscope. It is designed for high-resolution imaging of living specimens.

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Lab products found in correlation

Fluoromount g, by Southern Biotech (2 mentions) Cell navigator cell plasma membrane staining kit, by AAT Bioquest (2 mentions) Dykddddk flag tagged specific polyclonal alexa fluor 647 conjugated antibody, by Cell Signaling Technology (2 mentions) Dapi fluoromount g, by Southern Biotech (2 mentions) Gfr matrigel, by BD (1 mentions) Alexa fluor labeled secondary antibody, by Thermo Fisher Scientific (1 mentions) Type 1 collagen, by BD (1 mentions) Fluoromount g, by Leica (1 mentions) 4 6 diamidino 2 phenylindole dapi, by Abcam (1 mentions) Rhodamine conjugated phalloidin, by Thermo Fisher Scientific (1 mentions) Eclipse ni u microscope, by Nikon (1 mentions) Phosphate buffered saline (pbs), by Boston BioProducts (1 mentions) Vectashield with 4 6 diamidino 2 phenylindole dapi, by Vector Laboratories (1 mentions) Alexa 488 labeled phalloidin, by Thermo Fisher Scientific (1 mentions)

6 protocols using tcs sp5 multiphoton laser scanning confocal microscope

Immunofluorescence Imaging of Adherent and 3D Cells

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Cells were fixed with 4% paraformaldehyde (PFA)/phosphate-buffered saline (PBS), permeabilized with 0.2% Triton X-100 (TX100), washed, and incubated with the indicated primary antibody overnight at 4°C. Cells were incubated with Alexa Fluor–labeled secondary antibody (Alexa 488, 546, or 647) and phalloidin (Molecular Probes, Grand Island, NY). Coverslips were mounted onto glass slides with Fluoromount-G (Southern Biotech, Birmingham, AL).
For 3D immunofluorescence, cells were resuspended in GFR-Matrigel (BD Biosciences, San Jose, CA) for 45 min at 37°C before addition of complete medium. Cells were fixed using 4% PFA, permeabilized using 0.2% TX100, and incubated with primary antibodies overnight at 4°C with fluorescence-labeled secondary antibodies and phalloidin for 3 h at 37°C. A TCS SP5 multiphoton laser-scanning confocal microscope (Leica Microsystems, Buffalo Grove, IL) with a Leica HC PL APO 40X/1.25-0.75 oil CS objective lens generated Z-stack images (0.5-μm optical sections), and the Leica AFM acquisition/analytical software suite was used to construct 3D projections.
For 3D spheroid invasion, samples were fixed with 4% paraformaldehyde, permeabilized with 0.2% TX100, and incubated with phalloidin 3h at 37°C. Confocal microscopy was with a Leica 20× HCX PL APO CS Dry UV 0.70NA objective lens to generate Z-stack images (2.5 μm optical sections).

Arden J.D., Lavik K.I., Rubinic K.A., Chiaia N., Khuder S.A., Howard M.J., Nestor-Kalinoski A.L., Alberts A.S, & Eisenmann K.M. (2015). Small-molecule agonists of mammalian Diaphanous–related (mDia) formins reveal an effective glioblastoma anti-invasion strategy. Molecular Biology of the Cell, 26(21), 3704-3718.

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Immunofluorescence Imaging of Cells in 2D and 3D

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Cells imaged in 2D were fixed with 4% paraformaldehyde in PBS (PFA/PBS), permeabilized with 0.2% Triton X-100 and incubated with primary antibody overnight at 4°C. Cells were incubated with secondary antibody (Alexa 488, 546, or 647) and phalloidin (Molecular Probes) for 3h at 37°C. Coverslips were mounted with fluoromount-G (Southern Biotech).
For 3D IF, cells were mixed with 2mg/ml Type-I collagen (BD Biosciences), gelled for 1h at 37°C and incubated 24h. Cells were fixed, permeabilized and incubated with antibodies as above. Gels were covered with fluoromount-G.
Images were acquired using a TCS SP5 multiphoton laser scanning confocal microscope (Leica Microsystems) with 458, 488, 514, 561 and 633nm laser lines in a sequential manner. Z-stack images were acquired using 0.5µm optical sections, creating 3D reconstructions for image analysis using MetaMorph software.

Wyse M.M., Goicoechea S., Garcia-Mata R., Nestor-Kalinoski A.L, & Eisenmann K.M. (2017). mDia2 and CXCL12/CXCR4 Chemokine Signaling Intersect to Drive Tumor Cell Amoeboid Morphological Transitions. Biochemical and biophysical research communications, 484(2), 255-261.

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Visualization of FLAG-tagged mIR Expression in HEK-Cre+ Cells

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HEK-Cre+ cells and the 3×FLAG-mIR cell line were grown out in 24 well plates on glass slides. The HEK-Cre+ cells were transfected with the pPNTlox2 or the pPNTlox2-3×FLAG-mIR plasmid and stained 48 h post transfection. All samples were incubated with the plasma membrane dye, Cell Navigator^™ Cell Plasma Membrane Staining Kit (AAT Bioquest). Following plasma membrane staining, cells were fixed with 4% paraformaldehyde, blocked with 2% BSA, and stained with DYKDDDDK FLAG-tagged specific polyclonal Alexa Fluor^® 647 conjugated antibody (Cell Signaling Technology). Cells were mounted to glass microscope slides via DAPI Fluoromount-G^® (SouthernBiotech) and dried. Image analysis was carried out utilizing a Leica TCS SP5 multiphoton laser scanning confocal microscope (Leica Microsystems) using a 63 × 1.4 NA lens.

Morran M.P., Al-Dieri A.G., Nestor-Kalinoski A.L., Jordan R.K., Gupta N.K, & McInerney M.F. (2018). Insulin receptor based lymphocyte trafficking in the progression of type 1 diabetes. Journal of Biological Methods, 5(1), e85.

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Visualizing FLAG-Tagged Protein Expression in Cells

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HEK-Cre+ cells and the 3×FLAG-mIR cell line were grown out in 24 well plates on glass slides. The HEK-Cre+ cells were transfected with the pPNTlox2 or the pPNTlox2-3×FLAG-mIR plasmid and stained 48 h post transfection. All samples were incubated with the plasma membrane dye, Cell Navigator™ Cell Plasma Membrane Staining Kit (AAT Bioquest). Following plasma membrane staining, cells were fixed with 4% paraformaldehyde, blocked with 2% BSA, and stained with DYKDDDDK FLAG-tagged specific polyclonal Alexa Fluor^® 647 conjugated antibody (Cell Signaling Technology). Cells were mounted to glass microscope slides via DAPI Fluoromount-G^® (SouthernBiotech) and dried. Image analysis was carried out utilizing a Leica TCS SP5 multiphoton laser scanning confocal microscope (Leica Microsystems) using a 63 × 1.4 NA lens.

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Immunofluorescence Imaging of Kidney Cells

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Cryosections of kidney specimens or cultured cells were fixed, permeabilized, and stained with antibodies against indicated molecules followed by staining with a secondary antibody conjugated with Alexa Fluor 488 or 594 or stained with rhodamine-conjugated phalloidin (Invitrogen, IL, United States) followed by counterstaining with 4,6-diamidino-2-phenylindole (DAPI, Abcam). Images were documented by using the Nikon Eclipse Ni-U microscope (Nikon, Tokyo, Japan) and the Leica TCS SP5 multiphoton laser scanning confocal microscope (Leica Microsystems Inc., Buffalo Grove, IL, United States).

Chen B., Alam Z., Ge Y., Dworkin L, & Gong R. (2022). Pharmacological Melanocortin 5 Receptor Activation Attenuates Glomerular Injury and Proteinuria in Rats With Puromycin Aminonucleoside Nephrosis. Frontiers in Physiology, 13, 887641.

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Quantifying Cell Spreading Dynamics

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DU145 or PC3 (control or RNase L-knockdown) were plated on fibronectin-coated coverslips for indicated times up to 100 min. Attached and spread cells were fixed with 3.7% paraformaldehyde in PBS (Boston Bioproducts, Ashland, MA, USA) for 10 min and permeabilized with 0.1% Triton-X-100 for 5 min. F-actin was labeled with Alexa 488-labeled phalloidin (Life Technologies, CA, USA) and mounted in Vectashield with DAPI (4′,6-diamidino-2-phenylindole, Vector Laboratories, Burlingame, CA, USA). Cells were imaged by the use of a Leica TCS SP5 multiphoton laser scanning confocal microscope (Leica Microsystems, Weitzler, Germany), and cell area was quantitated using NIH Image J software. Experiments were repeated in triplicate with at least 30 measurements per time point for each experiment. Data are shown as mean area at indicated time points ± SEM.

Dayal S., Zhou J., Manivannan P., Siddiqui M.A., Ahmad O.F., Clark M., Awadia S., Garcia-Mata R., Shemshedini L, & Malathi K. (2017). RNase L Suppresses Androgen Receptor Signaling, Cell Migration and Matrix Metalloproteinase Activity in Prostate Cancer Cells. International Journal of Molecular Sciences, 18(3), 529.

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