Leica tcs sp5 confocal laser scanning microscope system

Manufactured by Leica Microsystems

Sourced in Germany, United States

The Leica TCS SP5 is a confocal laser scanning microscope system designed for advanced imaging applications. It features multiple laser excitation sources and a high-performance detection system to capture detailed, high-resolution images of biological and materials samples.

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Lab products found in correlation

Perfluorodecalin, by Merck Group (1 mentions) Volocity 6, by PerkinElmer (1 mentions) Nanozoomer s60 digital, by Hamamatsu Photonics (1 mentions) Ab150061, by Abcam (1 mentions) Ab53212, by Abcam (1 mentions) Af332, by R&D Systems (1 mentions) Alexa fluor 488 conjugated donkey anti rabbit igg h l, by Abcam (1 mentions) Nanozoomer s60 digital slide scanner, by Hamamatsu Photonics (1 mentions)

6 protocols using leica tcs sp5 confocal laser scanning microscope system

Confocal Microscopy Imaging of Leaf Samples

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Leaves were cut into small pieces and placed on glass slides with a drop of perfluorodecalin (Sigma-Aldrich, St Louis, MO) and observed with a Leica TCS SP5 confocal laser scanning microscope system (Leica Micro- systems, Bannockburn, IL, USA). GFP fluorescence was detected with excitation at 488 nm and emission capture at 500–530 nm. The DAPI was excited with a 405 nm laser, and emission was collected from 405–500 nm. SNARF-1 was excited at 514 nm and emission was collected from 600–700 nm. spRFP:AFVY was excited at 532 nm and emission was collected from 550–700 nm. Images were captured at 2,361 μm intervals using a × 20 objective Final figures were prepared using ImageJ software (imagej.nih.gov/ij/).

Bak A., Cheung A.L., Yang C., Whitham S.A, & Casteel C.L. (2017). A viral protease relocalizes in the presence of the vector to promote vector performance. Nature Communications, 8, 14493.

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Subcellular Localization of CsLBD Proteins

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Plant-mPloc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) was used to predict protein subcellular localization. The CsLBD sequences were amplified and cloned into the binary vector pCV-eGFP-N1 digested with Kpn I and BamH I (Supplementary Dataset 4). The recombinant binary constructs and pCV-eGFP-N1 (control) were individually introduced into Agrobacterium tumefaciens strain GV3101. Agrobacterium cultures carrying the recombinant vectors were grown overnight at 28 °C, cells were pelleted and resuspended in infiltration buffer (10 mM MgCl₂, 10 mM MES and 150 µM acetosyringone) with OD600 = 1. The cell suspensions were incubated at room temperature and then infiltrated into 4–6 week old N. benthaminan leaves. Expression of fluorescent proteins was observed with a Leica TCS SP5 confocal laser scanning microscope system (Leica Microsystems, Bannockburn, IL, USA) at 48 h post-agroinfiltration. Fluorescence of GFP was observed at 495–545 nm.

Zhang X., He Y., He W., Su H., Wang Y., Hong G, & Xu P. (2019). Structural and functional insights into the LBD family involved in abiotic stress and flavonoid synthases in Camellia sinensis. Scientific Reports, 9, 15651.

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Proinsulin and Insulin Expression in Pancreas

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Frozen pancreatic tissue sections were analysed through double immunofluorescence to evaluate the expression patterns of proinsulin and insulin. Primary antibodies Polyclonal Guinea Pig Anti-Human Insulin (cat. A0564, Agilent Technologies, Santa Clara, CA, USA) diluted 1:2000 and Mouse Monoclonal Anti-Human Proinsulin (cat. GS9A8–Developmental Study Hybridoma Bank, Iowa City, IA, USA) (epitope: B–C junction of proinsulin spanning aa 26–37) [13 (link)–15 (link)] diluted 1:100 in PBS 1X supplemented with 1% BSA overnight in a damp chamber at 4°C were used. Images were acquired using a Leica TCS SP5 confocal laser scanning microscope system (Leica Microsystems, Wetzlar, Germany) and analysed using Volocity 6.3 software (Perkin Elmer, Waltham, MA, USA). For detailed methods please refer to the ESM Methods section.

Brusco N., Sebastiani G., Di Giuseppe G., Licata G., Grieco G.E., Fignani D., Nigi L., Formichi C., Aiello E., Auddino S., Quero G., Cefalo C.M., Cinti F., Mari A., Ferraro P.M., Pontecorvi A., Alfieri S., Giaccari A., Dotta F, & Mezza T. (2022). Intra-islet insulin synthesis defects are associated with endoplasmic reticulum stress and loss of beta cell identity in human diabetes. Diabetologia, 66(2), 354-366.

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Quantifying Colocalization of MDA5 in Pancreatic Islets

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Images were acquired using Leica TCS SP5 confocal laser scanning microscope system (Leica Microsystems, Wetzlar, Germany) and NanoZoomer S60 Digital slide scanner (Hamamatsu Photonics K.K., Hamamatsu City, Japan). Images were analyzed using Leica Application Advance Fluorescence (LasAF) and with NDP view2 plus software. In particular, the percentage of colocalization rate of MDA5-insulin and MDA5-glucagon for each pancreatic islet was quantified determining the region of interest (ROI), drawn to calculate the "colocalization rate" (which indicates the extent of colocalization in percentage) as ratio between the colocalization area and the image foreground (in which the colocalization area represents the ratio of the area of colocalizing fluorescence signals and the image foreground represents the image area with fluorescence signal, calculated from the difference between the area of ROI and the area of image background).

Nigi L., Brusco N., Grieco G.E., Fignani D., Licata G., Formichi C., Aiello E., Marselli L., Marchetti P., Krogvold L., Dahl‐Jørgensen K., Sebastiani G., & Dotta F. (2021). Increased expression of viral sensor MDA5 in pancreatic islets and in hormone-negative endocrine cells in recent onset type 1 diabetic donors.

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Immunohistochemical Localization of TMEM16A and c-Kit

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Anal tissues were isolated, and the skeletal muscle fibers were removed, immediately followed by embedded in Optimal cutting temperature compound (Bio-Tek). Cryosections with a 10-μm thickness were fixed in pre-cooled acetone for 10 min and washed with PBS three times at room temperature. The nonspecific binding of primary antibodies was blocked by incubation with PBST (0.3% Triton in PBS) containing 1% BSA for 1 h. Incubation was carried out overnight at 4°C with a rabbit polyclonal antibody to TMEM16A (ab53212, 1:100; Abcam) and a goat polyclonal antibody against c-Kit (AF332, 1:20; R&D systems). The specificity of these antibodies has been established by vendors and others. After washing in PBS, cells were incubated with an Alexa Fluor 488-conjugated donkey anti-Rabbit IgG H&L (ab150061, 1:500; Abcam) or an Alexa Fluor 594-conjugated rabbit anti-goat IgG (H+L) (A27016, 1:500; Life tech) for 1 h. Negative controls were performed by omitting the primary antibodies. Immunoreactivity was evaluated using a Leica TCS SP5 confocal laser scanning microscope system (Leica Microsystems Inc., Buffalo Grove, IL).

Lu P., Chen J., Zhang C., Saur D., Baer C.E., Lifshitz L.M., Fogarty K.E, & ZhuGe R. (2021). Oscillating calcium signals in smooth muscle cells underlie the persistent basal tone of internal anal sphincter. Journal of cellular physiology, 236(8), 5937-5952.

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Quantifying Colocalization of MDA5 with Insulin and Glucagon

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Images were acquired using a Leica TCS SP5 confocal laser scanning microscope system (Leica Microsystems, Wetzlar, Germany) and a NanoZoomer S60 Digital slide scanner (Hamamatsu Photonics K.K., Hamamatsu City, Japan). Images were analyzed using Leica Application Advance Fluorescence (LasAF) and with the NDP view2 plus software. In particular, the percentage of colocalization rate of MDA5-insulin and MDA5-glucagon for each pancreatic islet was quantified determining the region of interest (ROI), drawn to calculate the “colocalization rate” (which indicates the extent of colocalization in percentage) as the ratio between the colocalization area and the image foreground (in which the colocalization area represents the ratio of the area of colocalizing fluorescence signals, and the image foreground represents the image area with fluorescence signal, calculated from the difference between the area of ROI and the area of the image background).

Nigi L., Brusco N., Grieco G.E., Fignani D., Licata G., Formichi C., Aiello E., Marselli L., Marchetti P., Krogvold L., Jorgensen K.D., Sebastiani G, & Dotta F. (2022). Increased Expression of Viral Sensor MDA5 in Pancreatic Islets and in Hormone-Negative Endocrine Cells in Recent Onset Type 1 Diabetic Donors. Frontiers in Immunology, 13, 833141.

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