Application suite 4

Manufactured by Leica

Sourced in Germany

Leica Application Suite 4.5 is a software application that enables the control and management of various Leica imaging equipment. It provides an integrated platform for image acquisition, processing, and analysis.

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Leica Application Suite (348 citations) Leica Application Suite version 4.4 (24 citations) LEICA Application Suite software, version 4.0 (10 citations) Leica Applications Suite (5 citations) Application Suite (LAS V4.4). (3 citations) Leica Application Suite (V4.4.0) (3 citations) Leica Application (2 citations) Leica Application Suite (4.12.0) (2 citations) Application Suite Image 4.0 System (2 citations) Leica Application Suite (LAS) Version 4.8.0 software (2 citations)

40 protocols using application suite 4

Immunohistochemical Analysis of Francisella Infection

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Immunohistochemistry (IHC) analysis was performed for qPCR-positive spleen and liver samples from four animals. These samples were classified as level 3 (n = 4, two spleens, two livers) or level 2 (n = 4, two spleens, and two livers). IHC was performed on 3 µm paraffin sections using Bond Polymer Refine Detection (#DS9800, Leica Biosystems, Newcastle, UK).
Deparaffinized slides were incubated in a citrate solution (pH = 6) for 20 min, at 100 °C for epitopes retrieval. Staining was performed using an F. tularensis LPS-directed mouse monoclonal antibody (FB11, used at 1:1000, ThermoFisher Scientific, Rockford USA) applied for one hour at room temperature. Peroxidase revelation was performed using the Bond Polymer Refine Detection Kit (Leica Biosystems, Newcastle, UK), according to the manufacturer's recommendations. The pictures were taken with a Leica DFC295 camera, using the Leica Application Suite 4.13.
The positive control was F. tularensis-infected amoebae fixed with 4% formaldehyde assembled in a cytobloc using Shandon reagent and paraffin-embedded. The negative control was Francisella philomiragia-infected amoebae treated at the same time and similarly to the positive control.

Ammam I., Brunet C.D., Boukenaoui-Ferrouk N., Peyroux J., Berthier S., Boutonnat J., Rahal K., Bitam I, & Maurin M. (2022). Francisella tularensis PCR detection in Cape hares (Lepus capensis) and wild rabbits (Oryctolagus cuniculus) in Algeria. Scientific Reports, 12, 21451.

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Gut Tissue Histomorphometric Analysis

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After 48 h in 4% formaldehyde, gut sections of ~3 mm were cut for tissue embedding cassettes and stored in 70% ethanol for 5 days. Afterwards, the samples were embedded in paraffin wax blocks. Sections of 5 µm were processed using a microtome (Leica RM2255, Leica Biosystems GmbH, Nussloch, Germany), mounted on glass slides, and stained (Leica Auto-Stainer XL ST5010, Leica Biosystems GmbH, Nussloch, Germany) following the standard protocol for Alcian blue periodic acid–Schiff (AB–PAS) reaction. The measurements for intestinal morphology were taken using a light microscope (Leica DM 6000 B, Leica, Darmstadt, Germany) and the image processing software Leica Application Suite 4.13 (Leica Application Suite 4.13, Darmstadt, Germany). All examined morphometric indices were made on six well-orientated villi and crypts. Besides length, width, and area measurements of villi and crypts, tunica muscularis thickness (circular and longitudinal muscle layers together) was determined in six randomly selected points. The villus height-to-crypt depth ratio (VC ratio) was also calculated. Goblet cells were counted in six villi and crypts.

Rajković E., Schwarz C., Kapsamer S.B., Schedle K., Reisinger N., Emsenhuber C., Ocelova V., Roth N., Frieten D., Dusel G, & Gierus M. (2022). Evaluation of a Dietary Grape Extract on Oxidative Status, Intestinal Morphology, Plasma Acute-Phase Proteins and Inflammation Parameters of Weaning Piglets at Various Points of Time. Antioxidants, 11(8), 1428.

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Seed Coat Colour Analysis

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Seed coat colour measurements were done on samples of harvested seeds using a stereomicroscope integrated with a computer software (Leica Application Suite 4.0, South Africa). Measurements of the colour codes red, green and blue (RGB) were recorded and converted to hue, saturation and lightness (HSL), Bautista et al. (2014) .

Mandizvo T, & Odindo A.O. (2019). Seed coat structural and imbibitional characteristics of dark and light coloured Bambara groundnut (Vigna subterranea L.) landraces. Heliyon, 5(2), e01249.

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Trichome Density and Length Analysis

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The methodology used to identify and count trichomes was fully described by [31 (link)]. The fifth leaf of each plant was dissected into 15 × 3 mm strips of the leaf blade for the analysis. The samples were affixed to the edge of microscope slides using transparent nail polish. A support made of Styrofoam with a slit was used to secure the slides horizontally but elevated from the stage to keep the trichomes perpendicular to the objective-stage direction. Photographs were taken using a stereomicroscope Leica S8AP0 (Wetzlar, Germany) at 50× magnification coupled to a Leica DFC295 camera (Wetzlar, Germany). The lateral views of the trichomes allowed the correct measurement, classification, and counting of each type. Eight individual plants per genotype were sampled, and four different strips were analyzed per plant for each leaf side (abaxial/adaxial). From the pictures, trichome counting was performed for density estimation. Trichome length measurements were performed using the manufacturer’s analytical software (Leica Application Suite 4.0, Wetzlar, Germany).

Vendemiatti E., Therezan R., Vicente M.H., Pinto M.D., Bergau N., Yang L., Bernardi W.F., de Alencar S.M., Zsögön A., Tissier A., Benedito V.A, & Peres L.E. (2022). The Genetic Complexity of Type-IV Trichome Development Reveals the Steps towards an Insect-Resistant Tomato. Plants, 11(10), 1309.

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Aorta Histology and Immunohistochemistry

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Murine and human aortas were fixed in 4% paraformaldehyde embedded in paraffin. Four ‐micrometers thick cross sections were prepared for hematoxylin and eosin stain and Elastica‐van Gieson staining for morphological assessment. For immunohistochemistry, rabbit anti‐VPO1 antibody VPO1 (5 μg/mL; EMD Millipore, USA), rabbit anti‐3‐Cl‐tyr antibody (2.5 μg/mL; Cell Science, USA), mouse anti‐KLF4 antibody (5 μg/mL; Abcam, UK), rabbit anti‐α−SMA and SM‐22α antibodies (1.25 μg/mL; Sigma, USA), and rabbit anti‐MMP‐2 antibody (2.5 μg/mL; Abcam, UK) were used. Paraffin sections were rehydrated and endogenous peroxidase activity was blocked for 30 minutes in methanol containing 0.3% hydrogen peroxide. Five percent normal goat serum (Sigma‐Aldrich, St. Louis, MO, USA) was incubated for 30 minutes at room temperature to block non‐specific background staining. Primary antibodies were incubated at 4°C overnight, followed by 60 minutes in biotinylated secondary antibody (2 μg/mL; Abcam, England). All specimens were counterstained with hematoxylin staining solution (Beyotime Institute of Biotechnology, China). Sections were scanned using OLYMPUS CX41 and Leica Application Suite 4.0 software. Morphological analysis and collateral degree was determined.

Peng H., Zhang K., Liu Z., Xu Q., You B., Li C., Cao J., Zhou H., Li X., Chen J., Cheng G., Shi R, & Zhang G. (2018). VPO1 Modulates Vascular Smooth Muscle Cell Phenotypic Switch by Activating Extracellular Signal‐regulated Kinase 1/2 (ERK 1/2) in Abdominal Aortic Aneurysms. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 7(17), e010069.

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Quantifying Collateral Arteries and Angiogenesis

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The gastrocnemius muscles were harvested at day 14 after femoral artery ligation. The midzone of the muscles (the 5 mm wide centermost section) was trimmed. The samples were embedded in paraffin, and 4 μm thick cross-sections were made to prepare for hematoxylin and eosin (H&E) staining. The number of collateral arteries per field was measured under a microscope at 40x magnification. For immunohistochemistry, we used antibodies against CD31 (1 : 200, Abcam, UK). The paraffin section was rehydrated, and endogenous peroxidase activity was blocked for 30 min in methanol containing 0.3% hydrogen peroxide. The section was incubated with the primary antibody at 4°C overnight, followed by 60 min of incubation with the biotinylated secondary antibody (1 : 500, Abcam, UK) [36 (link)]. All specimens were counterstained with hematoxylin staining solution (Beyotime Institute of Biotechnology, China) and then sealed with neutral gum for storage. Analyses of morphology and degree of angiogenesis were performed after scanning the section through OLYMPUS CX41 and Leica Application Suite 4.0 software.

Wu S.H., Zhang F., Yao S., Tang L., Zeng H.T., Zhu L.P, & Yang Z. (2020). Shear Stress Triggers Angiogenesis of Late Endothelial Progenitor Cells via the PTEN/Akt/GTPCH/BH4 Pathway. Stem Cells International, 2020, 5939530.

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Seed Color Measurements in Bambara Groundnut

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Seed coat color measurements were performed on Bambara groundnut genotypes using a stereomicroscope integrated with computer software (Leica Application Suite 4.0, South Africa). Hue, saturation, and lightness measurements were recorded and converted from red, green, and blue (RGB) codes (HSL) [12 (link)].

Kunene S., Odindo A.O., Gerrano A.S, & Mandizvo T. (2022). Screening Bambara Groundnut (Vigna subterranea L. Verdc) Genotypes for Drought Tolerance at the Germination Stage under Simulated Drought Conditions. Plants, 11(24), 3562.

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Morphological Study of Chinese Moths

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Specimens were collected in China since 1998 using light traps. Wingspan was measured from the tip of the left forewing to the tip of the right forewing. Genitalia slides were prepared following the methods introduced by Li (2002) . All images were captured with digital microscopes (Leica M205A and Leica DM750), coupled with the Leica Application Suite 4.2 software. Terminology follows Gozmány (1978) . The male and female genitalia are described from the ventral view.
All the specimens examined, including the type series of the new species, are deposited in the Insect Collection of Nankai University, Tianjin, China (NKU).

Yu S, & Wang S. (2023). Opacoptera Gozmány (Lepidoptera, Lecithoceridae) from China, with descriptions of four new species. ZooKeys, 1158, 133-146.

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Genitalia Dissection of Thai Specimens

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The examined specimens were collected from Thailand in 1984, and were borrowed from the Natural History Museum of Denmark, where all types are deposited. Genitalia dissection and mounting methods follow the methods introduced by Li (2002) . Photographs of adults were taken with a Leica M205A stereomicroscope plus Leica Application Suite 4.2 software, and illustrations of genitalia were prepared using a Leica DM750 microscope.

Yin A, & Wang S. (2016). Taxonomic study of the genus Meleonoma Meyrick from Thailand (Lepidoptera, Gelechioidea). ZooKeys.

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Insect Collection Protocols at Nankai University

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The present study is based on material deposited in the Insect Collection, College of Life Sciences, Nankai University (NKU), Tianjin, China.
Adults were examined with an Olympus SZX16 stereo microscope. Male and female genitalia were prepared using the unrolling technique as described by Pitkin (1986) and Huemer (1988) or follow the methods introduced by Li (2002) . Images of adults and genitalia were taken with a Leica M205A stereo microscope and Leica DM750 microscope, coupled with the Leica Application Suite 4.2 software, respectively.
Biological data were mainly extracted from bibliographic sources. The distribution of species was established primarily from the material examined and supplemented by data in the literature. The “Material” is arranged in geographical order from northwest to southeast and countries referred to by their current names. The type material of the new described taxa is deposited in NKU. The descriptive terminology of the genitalia structures generally follows Huemer and Karsholt (2010) (link).

Li H, & Bidzilya O. (2019). New species and new records of the genus Scrobipalpa Janse (Lepidoptera, Gelechiidae) from China. ZooKeys, 840, 101-131.

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