Leica tcs sp5 confocal microscope

Manufactured by Leica camera

Sourced in Germany, United States, Canada

The Leica TCS SP5 is a confocal microscope designed for high-performance imaging. It utilizes advanced laser technology and optical systems to enable detailed, high-resolution imaging of samples. The TCS SP5 is capable of capturing three-dimensional images with exceptional clarity and precision.

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143 protocols using leica tcs sp5 confocal microscope

Immunofluorescence Imaging of Adherent Cells and Vascular Organoids

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Adherent cells: Indirect immunofluorescence on adherent cells was performed as previously described (Bellou, 2012 #68) using primary and secondary antibodies (listed in Materials and Methods Supplementary). Cell nuclei were stained using propidium iodide-PI (Sigma), samples were mounted in moviol-dabco, and images of nine fields were taken on a Leica TCS SP5 confocal microscope using HCX PL APO CS 40 × 1.25 OIL objective.
Vascular organoids/spheroids: Vascular organoids or spheroids consisting of 1,000 cells/spheroid were fixed in 3.7% paraformaldehyde for 1 h at RT, permeabilized with 0.2% Triton-X/0.9% gelatin solution for 1 h, and 0.5% Triton-X/0.9% gelatin solution for 15 min, and incubated with primary antibodies overnight at 4°C (Supplementary Table S2). Next day, the vascular organoids were washed 5x with 0.2% Triton-X and incubated with secondary antibodies for 1 h (Supplementary Table S2). After rinsing 5x with 0.2% Triton-X and incubation with Draq5 (Thermo Fisher Scientific) for 10 min, images were taken on a Leica TCS SP5 confocal microscope using HCX PL APO CS 40 × 1.25 OIL objective. At least 10 vascular organoids or spheroids were analyzed per experiment.

Markou M., Kouroupis D., Badounas F., Katsouras A., Kyrkou A., Fotsis T., Murphy C, & Bagli E. (2020). Tissue Engineering Using Vascular Organoids From Human Pluripotent Stem Cell Derived Mural Cell Phenotypes. Frontiers in Bioengineering and Biotechnology, 8, 278.

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Measuring Intracellular ROS and SOD Activity

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The cells were loaded with 10 μM H2DCF-DA (Invitrogen, Molecular Probes, USA) or 10 μM dihydroethidium (DHE, Invitrogen, Molecular Probes, USA) at 37°C for 30 minutes according to the manufacturer's instructions. After removing excess probes, the cells were kept at 37°C containing 5% CO₂. Fluorescence intensity was detected by Leica TCS SP5 confocal microscope (Leica, Germany). For each sample 10,000 events were collected. For lung tissues, add 0.1% ROS (DHE) probe about 10 edged up on the lung tissue of frozen section, after incubation for 30 minutes at 37°C, washing 2-3 times with phosphate-buffered saline (PBS) and observation by Leica TCS SP5 confocal microscope. Superoxide dismutases (SOD) activity was measured using the SOD assay kit WST (Nanjing jiancheng bioengineering institute, china). The ELISA kits of TNF-α and IL-6 were purchased from R&D Systems (Minneapolis, MN).

Chen Y., Luo G., Yuan J., Wang Y., Yang X., Wang X., Li G., Liu Z, & Zhong N. (2014). Vitamin C Mitigates Oxidative Stress and Tumor Necrosis Factor-Alpha in Severe Community-Acquired Pneumonia and LPS-Induced Macrophages. Mediators of Inflammation, 2014, 426740.

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Immunohistochemistry and TUNEL Assay for Apoptosis Analysis

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Immunohistochemistry for cleaved caspase-3 was conducted using an anti-rat cleaved caspase-3 antibody (Abcam, Cambridge, UK) and a secondary polyclonal antibody to rabbit IgG coupled with FITC (Abcam, Cambridge, UK). Fluorescence images were captured with a Leica TCS-SP5 confocal microscope (Leica, Wetzlar, Germany) and processed with Leica X software (Version 3.7.4). Apoptosis in infarcted brain tissue was analyzed using an In Situ Cell Death Detection Kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Fluorescence from TUNEL stained signals was captured with a Leica TCS-SP5 confocal microscope and processed with Leica X software (Version 3.7.4). Neurological deficits were evaluated as previously described [11 (link)].

Chung K., Ullah I., Yi Y., Kang E., Yun G., Heo S., Kim M., Chung S.E., Park S., Lim J., Lee M., Rhim T, & Lee S.K. (2024). Intranasal Delivery of Anti-Apoptotic siRNA Complexed with Fas-Signaling Blocking Peptides Attenuates Cellular Apoptosis in Brain Ischemia. Pharmaceutics, 16(2), 290.

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Confocal Imaging of Cells and Tissues

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Images (1024 × 1024 pixels) were taken on a TCS SP5 Leica confocal microscope with a HCX PL APO CS N.A.1.25 × 40 oil immersion objective using Leica LAS‐AF software. For large tissue areas, multiple images were taken using a motorised XY stage and then stitched together using the LAS‐AF software. For cell spacing, single confocal planes were analysed. For nucleus–Golgi angles, image stacks were taken with confocal sections at 0.17 μm intervals.

Brock L.J., Economou A.D., Cobourne M.T, & Green J.B. (2015). Mapping cellular processes in the mesenchyme during palatal development in the absence of Tbx1 reveals complex proliferation changes and perturbed cell packing and polarity. Journal of Anatomy, 228(3), 464-473.

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Confocal Microscopy Imaging Protocol

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Samples were imaged using a TCS SP5 Leica Confocal microscope with a 40× oil (NA of 1.3), 63× oil (NA of 1.4) or a 40× water (NA 1.2) objective. The following figures were deconvolved using Huygens software: Figs 1A,D, 3B, 5D.

Redl S., de Jesus Domingues A.M., Caspani E., Möckel S., Salvenmoser W., Mendez-Lago M, & Ketting R.F. (2021). Extensive nuclear gyration and pervasive non-genic transcription during primordial germ cell development in zebrafish. Development (Cambridge, England), 148(2), dev193060.

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FRAP Analysis of Granule Dynamics

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FRAP was performed on a TCS SP5 Leica confocal microscope, equipped with a FRAP-booster, using a 63x oil objective with an NA of 1.4 (BmN4 cells) or a 63x water objective with an NA of 1.2 (Bb). In BmN4 cells, entire granules were bleached in a fixed region of 1.5μm ø and recovery was followed for 1500 frames (0.2s/frame). Bbs were bleached partially in a fixed region of 2.5μm ø and recovery was followed for 1500 frames (0.5s/frame). Regions (pre-- and post-bleach) were tracked using TrackMate (Tinevez et al., 2017 (link)). 10 pre-bleach frames were recorded and after background subtraction, the average intensity was used as pre-bleach intensity. Post-bleach frames were background subtracted and to make replicates comparable, post-bleach frame #1 of each measurement was set to 0 and corresponding pre-bleach intensity was corrected for this. Normalization of Buc-eGFP and mCherry-Tdrd6a was performed by plotting intensities of mCherry-eGFP using the same microscope settings as for the FRAP. Intensities were plotted after background subtraction and the resulting curve was used to calculate protein ratios using the initial/pre-bleach intensities of each experiment.

Roovers E.F., Kaaij L.J., Redl S., Bronkhorst A.W., Wiebrands K., de Jesus Domingues A.M., Huang H.Y., Han C.T., Riemer S., Dosch R., Salvenmoser W., Grün D., Butter F., van Oudenaarden A, & Ketting R.F. (2018). Tdrd6a Regulates the Aggregation of Buc into Functional Subcellular Compartments that Drive Germ Cell Specification. Developmental Cell, 46(3), 285-301.e9.

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Histological Analysis of BAT and WAT

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Brown adipose tissue (BAT) and inguinal WAT (ingWAT) samples were fixed in 4% paraformaldehyde (PFA), dehydrated, and embedded in paraffin wax. The paraffin-embedded tissues were cut into 5-μm-thick sections and mounted on slides. The sections were then stained with primary and secondary antibodies according to the manufacturers’ instructions. Data were collected using H&E-stained sections from 4–6 mice in each group (20× and 40× magnifications). UCP1 immunohistochemistry was performed using a polyclonal anti-UCP-1 antibody (1:50). The sections were examined by microscopy (Olympus BX61/DP70). An immunofluorescence analysis of ingWAT was performed using anti-CD137 (1:100) and anti-Ki-67 (1:100) antibodies as well as anti-CD137 (1:100) and anti-UCP1 (1:50) antibodies. All the primary antibodies were incubated overnight at 4 °C. All the sections were incubated with the relevant secondary antibodies (1:250) and DAPI (1:500) for 2 h in the dark at room temperature. All the micrographs were obtained using a TCS SP5 Leica confocal microscope (40× magnification).

Igarashi Y., Nawaz A., Kado T., Bilal M., Kuwano T., Yamamoto S., Sasahara M., Jiuxiang X., Inujima A., Koizumi K., Imura J., Shibahara N., Usui I., Fujisaka S, & Tobe K. (2018). Partial depletion of CD206-positive M2-like macrophages induces proliferation of beige progenitors and enhances browning after cold stimulation. Scientific Reports, 8, 14567.

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Immunofluorescence Quantification of Stem Cell Markers

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In preparation for immunofluorescence staining, TS were dropped on poly-lysine coated slides (Thermo Fisher Scientific) and fixed in 4% PBS paraformaldehyde. Immunofluorescence was performed as previously described [23 (link)], cells were incubated with SOX-2 or Nestin (Millipore) primary antibodies, followed by fluorescent conjugated secondary antibodies (Life Technologies) and mounted with DAPI-containing mounting medium (Life Technologies). Confocal images were acquired on the TCS/SP5 Leica confocal microscope (Institut Cochin, Paris).
To calculate the total corrected fluorescence (TCF) of SOX-2 and Nestin we used ImageJ (v1.48, NIH). An outline was drawn around each TS and area, mean fluorescence and several adjacent background readings measured. The total corrected cellular fluorescence (TCCF) = integrated density – (area of selected cell × mean fluorescence of background readings), was calculated as previously described [55 ].

Harford-Wright E., Bidère N, & Gavard J. (2016). β-escin selectively targets the glioblastoma-initiating cell population and reduces cell viability. Oncotarget, 7(41), 66865-66879.

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Whole-Mount and Cryosection Immunostaining of Cardiobundles

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Immunostaining of cardiobundles was performed as previously described. Briefly, cardiobundles were fixed in 2% paraformaldehyde in PBS at 4°C overnight and used for whole-mount staining or snap-frozen in Tissue-Tek OCT (Sakura FineTek USA, Torrance, CA) for staining of cryosections. For whole-mount stains, tissues were incubated at 4°C overnight in 0.5% Triton X-100, 5% chicken serum, and 2.5% BSA in PBS and incubated with primary antibodies diluted in the blocking solution at 4°C for 12 hours (Supp. Table 1). Alexa Flour 488- and Alexa Flour 594-conjugated secondary antibodies (Thermo Fisher, Waltham, MA), DAPI, and Alexa Flour 647-conjugated phalloidin (Thermo Fisher, Waltham, MA) were applied at 4°C overnight. For cross-sectional stains, OCT-embedded cardiobundles were cut into 10-μm-thick sections, incubated for 90 min with blocking solution (with 0.2% Triton X-100), followed by 90 min incubation with secondary antibodies. Whole-mount tissues or cross-sections were mounted with Fluoromount-G (SoutherBiotech, Bringham, AL) and imaged with a TCS SP5 Leica confocal microscope.

Zhan R.Z., Rao L., Chen Z., Strash N, & Bursac N. (2021). Loss of Sarcomeric Proteins via Upregulation of JAK/STAT Signaling Underlies Interferon-γ-induced Contractile Deficit in Engineered Human Myocardium. Acta biomaterialia, 126, 144-153.

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Immunofluorescence Staining of IFITM3 and LAMP1

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3x10⁴ to 5x10⁴ THP-1 or CBCD34⁺ cells were seeded in Multitest slide glass 10-well 8mm (from MP Biomedicals) precoated with poly-L-lysine solution (Sigma-Aldrich) for 20 minutes. Cells were fixed with 4% paraformaldehyde (in 1X PBS) for 20 minutes at room temperature and permeabilized with 0.1% Triton X-100 for 20 minutes at room temperature. For blocking non-specific sites cells were incubated 30 minutes in PBG (5% BSA, 2% gelatin from cold water fish skin, from Sigma-Aldrich) and then stained for 2 till 16 hours with rabbit anti-IFITM3 polyclonal antibody (1:200 dilution) and or with a mouse monoclonal anti-LAMP1 (1:100, RRID:AB_470708). After 3 washes with 1X phosphate-buffered saline, cells were incubated with donkey anti-Rabbit IgG, Alexa Fluor 488 (RRID:AB_141708) or 555 (RRID:AB_162543) or 647 (RRID:AB_2536183) (1:500 dilution from Thermo Fisher Scientific) for 2 hours at room temperature. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) for 10 minutes at room temperature. Images were recorded using the TCS SP5 Leica confocal microscope, 60x with oil and quantified as integrated density with ImageJ software.

Petrillo C., Thorne L.G., Unali G., Schiroli G., Giordano A.M., Piras F., Cuccovillo I., Petit S.J., Ahsan F., Noursadeghi M., Clare S., Genovese P., Gentner B., Naldini L., Towers G.J, & Kajaste-Rudnitski A. (2018). Cyclosporine H Overcomes Innate Immune Restrictions to Improve Lentiviral Transduction and Gene Editing In Human Hematopoietic Stem Cells. Cell Stem Cell, 23(6), 820-832.e9.

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