Application suite version 3

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Application Suite version 3 is Leica's latest software package designed for lab equipment. It provides a comprehensive suite of tools for data analysis, visualization, and processing. The core function of this software is to enable efficient and streamlined management of experimental data and laboratory workflows.

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Application Suite X (LAS X) Version 3.5.5 Application Suite X software, version 3.7.0 Leica Application Suite Software (version 3.8 Leica Application Suite (LAS) version 3.8.0 Application Suite 3.3 Leica Applications Suite Leica Application Suite Leica Application

25 protocols using application suite version 3

Histological Analysis of Atherosclerotic Lesions

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En face and histological assessment of atherosclerotic lesion size has been described (11) (link). For histological aortic root analysis, frozen 5-μm sections from the aortic valve plane in 50-μm intervals were stained with Oil Red O staining with hematoxylin and light green counterstain. Picrosirius red stain and Masson’s trichrome stain with hematoxylin and eosin counterstain were used for assessment of fibrosis. For immunofluorescence, polyclonal anti-mouse CD3 (Dako A0452), anti-mouse B220 (RA3-6B2), anti-mouse CD11b (M1/70), anti-mouse CD11c (N418), polyclonal anti-mouse α-smooth muscle actin (ab15734) (Abcam, Cambridge, United Kingdom, and BioLegend), and the following secondary antibodies were used: donkey–anti-rat–IgG–AF488 (Invitrogen, Carlsbad, California), goat–anti-hamster–Cy3 (Jackson ImmunoResearch, Newmarket, United Kingdom), and donkey–anti-rabbit–IgG-AF555 (Life Technologies A31572, Life Technologies, Carlsbad, California). Images were obtained with a Leica DMI600B or DMI3000B microscope with 5×, 10×, and 20× original magnification using Leica Application Suite version 3.5.0 (Leica, Wetzlar, Germany). Analysis was conducted with NIH ImageJ and GIMP (version 2.8).

Nordlohne J., Helmke A., Ge S., Rong S., Chen R., Waisman A., Haller H, & von Vietinghoff S. (2018). Aggravated Atherosclerosis and Vascular Inflammation With Reduced Kidney Function Depend on Interleukin-17 Receptor A and Are Normalized by Inhibition of Interleukin-17A. JACC: Basic to Translational Science, 3(1), 54-66.

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Karyological Analysis of Cultivated Plants

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For karyological analysis seedlings and root meristems of the cultivated plants from the selected localities were used (for details see Appendix 1). For the pretreatment, the root tips were transferred to 0.002 M aqueous solution of 8-hydroxyquinoline at the temperature of 4°C for 16 hours. Then the root tips were fixed for at least 1 hour in acetic ethanol (glacial acetic acid and 96 % ethanol in the ratio 1: 3), washed in distilled water and hydrolyzed for 3 minutes in 1N HCL at 60°C, then washed in distilled water. The meristems were squashed using a cellophane technique (Murín 1960) and stained in 7 % Giemsa stain solution in Sörensen phosphate buffer for 3 hours. The slides were then washed in distilled water, dried, and observed in a drop of immersion oil. Selected c-metaphase plates were photographed (using a Leica DM 2500 microscope equipped with camera DFC 290 HD and software Leica application suite version 3.5.0, Switzerland) and number of chromosomes was determined.

Fráková V., Koprivý L., Mártonfiová L., Kocová V., Dudáš M., & Mártonfi P. (2021). Genome size, chromosome counts and distribution of Homogyne alpina (Asteraceae) in the Slovak Carpathians. THAISZIA - JOURNAL OF BOTANY, 31(2).

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In Vitro Tubule Formation Assay

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A Matrigel matrix (10 mg/mL) was gently pipetted into the bottom of each well of a 96-well plate. The plate was placed at 37 °C for 40 min whilst the Matrigel polymerised. Then, 4 × 10⁴ cells (and treatments/control reagents where appropriate) were plated on top of the Matrigel in each well in triplicate and incubated at 37 °C, 5% CO₂ and 95% humidity. Images of each well were captured at X5 magnification and intervals ranging from 30 to 1170 min using a Leica DMi1 microscope equipped with an MC120 HD camera and Leica Application Suite version 3.0.0 software (Leica Microsystems, Milton Keynes, UK). Analysis of the images was carried out using ImageJ, where the lengths of cell structures that form part of a tubule structure were measured to track the progress of tubule formation.

Frugtniet B.A., Ruge F., Sanders A.J., Owen S., Harding K.G., Jiang W.G, & Martin T.A. (2023). nWASP Inhibition Increases Wound Healing via TrKb/PLCγ Signalling. Biomolecules, 13(2), 379.

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Matrigel Tubule Formation Assay for Angiogenesis

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A Matrigel tubule formation assay was used to assess the angiogenic potential of human endothelial cells following SOCS-3 and SOCS-4 manipulation. This methodology was adapted from a previously described method^{45 (link)}. In brief, 50 µl of Matrigel matrix was gently pipetted into the bottom of each well in 96-well plate and incubated until polymerisation had occurred. Subsequently, cells were seeded onto the gel at a density of 40,000 cells/well and the plate was incubated at 37 °C, 5% CO₂ and 95% humidity. The plate was removed from the incubator and images were captured using a Leica DMi1 microscope equipped with a MC120 HD camera and Leica Application Suite version 3.0.0 software (Leica Microsystems, Milton Keynes, UK) at the 10^th hour. The total perimeter of each formed tubule in every image was measured using Image J.

Feng Y., Sanders A.J., Morgan L.D., Owen S., Ruge F., Harding K.G, & Jiang W.G. (2017). In vitro significance of SOCS-3 and SOCS-4 and potential mechanistic links to wound healing. Scientific Reports, 7, 6715.

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Intestinal Morphometric Analysis Protocol

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The procedure for intestinal morphometric analysis was as described by Oladokun et al. [48 (link)]. Briefly, fixed intestinal tissues were embedded in paraffin, sectioned (0.5 μm thick), and stained with hematoxylin and eosin for morphological examinations. In each cross-sectioned tissue, ten morphometric measurements including the villus height (from the base of the intestinal mucosa to the tip of the villus excluding the intestinal crypt), villus width (halfway between the base and the tip), crypt depth (from the base upward to the region of transition between the crypt and villi) [53 (link)] per slide were carried out using Leica 1CC50 W microscope at 4× Magnification (Leica Microsystems, Wetzlay, Germany) and an image processing and analysis system (Leica Application Suite, Version 3.4.0, Leica Microsystems, Wetzlay, Germany). The total mucosa thickness (villus height + crypt depth) was subsequently calculated from the obtained data.

Oladokun S., MacIsaac J., Rathgeber B, & Adewole D. (2021). Essential Oil Delivery Route: Effect on Broiler Chicken’s Growth Performance, Blood Biochemistry, Intestinal Morphology, Immune, and Antioxidant Status. Animals : an Open Access Journal from MDPI, 11(12), 3386.

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Intestinal Morphometric Analysis Protocol

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The procedure for intestinal morphometric analysis has previously been reported by Oladokun et al. (2021a) . Briefly, fixed intestinal tissues were embedded in paraffin, sectioned (0.5 μm thick), and stained with hematoxylin and eosin for morphological examinations. In each cross-sectioned tissue, ten morphometric measurements including the villus height (from the base of the intestinal mucosa to the tip of the villus excluding the intestinal crypt), villus width (halfway between the base and the tip), crypt depth (from the base upward to the region of transition between the crypt and villi) (Ozdogan et al., 2014 (link)) per slide were carried out using Leica 1CC50 W microscope at 4 × Magnification (Leica Microsystems, Wetzlay, Germany) and an image processing and analysis system (Leica Application Suite, Version 3.4.0, Leica Microsystems, Wetzlay, Germany).

Oladokun S, & Adewole D. (2023). The effect of Bacillus subtilis and its delivery route on hatch and growth performance, blood biochemistry, immune status, gut morphology, and microbiota of broiler chickens. Poultry Science, 102(4), 102473.

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Glycerin-based Fixation and Microscopic Analysis of EPNs

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To observe the EPNs in detail, they were evaluated after they were fixed using heat. A nematode was placed on a slide and different concentrations of glycerin and water were added in the following order: 1/20, 1/15, 1/10, 1/5 and 1/1. The different concentrations were added to the slides, which were placed on a heating plate.
When these processes were complete, the EPNs were observed under an optical microscope (Leica DM500, St. Gallen, Switzerland), and each stage was analyzed by measuring their length and width using Leica Application Suite, version 3.4.0 [23 ].

Guadarrama-Avila T.M., Ramírez-Trujillo J.A., Rodríguez-Ocampo T.G., Peña-Chora G., Arenas-Sosa I, & Hernández-Velázquez V.M. (2023). In Vivo Production, Development and Storage of Oscheius myriophila (Nematoda: Rhabditida) in Galleria mellonella (Lepidoptera: Pyralidae). Microorganisms, 11(10), 2571.

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Plant Morphometric Measurement Protocol

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Plant height was measured with a digital caliper. Phloem and xylem thickness, stem radius were measured using Leica application suite version 3.4.0 in all three replications. Although stem diameter was relatively preferred, we preferred to measure stem radius both vertical and horizontal in order to minimize measuring faults stemming from deformity in stem shape. Then average of four measurements were accepted as the stem radius in all replications.

KÜPLEMEZ H., & Yıldırım M.U. (2020). Effects of Cytokinin and Auxin on Plant Development and Vascular Tissues in Lens culinaris. Commagene Journal of Biology.

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Xenograft Growth Characterization in NSG Mice

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All animal procedures were approved by the Institutional Animal Care and Use Committees of the Ohio State University and Northwestern University. The procedure for the preparation of cell grafts and subrenal grafting were described previously (15 (link),18 (link)). Briefly, at the time of grafting, host NSG mice (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ, Jackson Laboratory) were ovariectomized and subcutaneously implanted with 70 mg slow-releasing hormone pellets. When the tumor volume of E2+P4 group was less than 1.0 mm³ at the endpoint (6 or 8 weeks after grafting), the LM was categorized as growth negative and removed from the analyses. Images of PTDXs on the kidney were captured from 3 directions (x, y and z axes) using a dissecting microscope connected to a computer with Leica Application Suite version 3.8 software (Leica Microsystems). The tumor volume was calculated by π/6× length × width × height. The average value of 3 to 9 xenografts per group in each experiment was considered as a single measurement. To study the growth control of SMCs and non-SMCs in MED12-LMs, PDTXs were prepared from a LM carrying a c.131G>A mutation in MED12, and the value of each xenograft (n ≥3) were considered as a single measurement.

Wu X., Serna V.A., Thomas J., Qiang W., Blumenfeld M.L, & Kurita T. (2017). Subtype-specific cancer-associated fibroblasts contribute to the pathogenesis of uterine leiomyoma. Cancer research, 77(24), 6891-6901.

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Immunofluorescent Detection of SARS-CoV-2 in DSAEK Grafts

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Virus-infected DSAEK grafts were fixed in 4% paraformaldehyde for 24 h and then permeated with 0.1% PBST. After extensive washing, non-specific immunoglobulin binding was blocked with 10% normal goat serum/1xPBS for 1 h at room temperature. The tissues were incubated overnight with the anti-SARS-CoV2 nucleocapsid antibody (1:500) followed by Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:800). Cell nuclei were counterstained with Hoechst 33258 for visualization. All images were taken with a Leica DM 4000B fluorescence microscope using Leica Application Suite version 3.8 (Leica Microsystems, Buffalo Grove, IL).

Krishna V.D., Roehrich H., Schroeder D.C., Cheeran M.C., Yuan C, & Hou J.H. (2022). In vitro infection of human ocular tissues by SARS-CoV-2 lineage A isolates. BMC Ophthalmology, 22, 518.

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