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  • Chronic Inflammation Under the Microscope

    In the course of chronic inflammation certain body areas are recurrently inflamed. This goes along with many human diseases. With the help of widefield light microscopy, the underlying processes can be examined from a cellular level to whole organisms. This article presents several widefield microscopy applications such as immunofluorescence, live-cell imaging, histology, and ratiometric analysis to get insight into the development of chronic inflammation, the related diseases, and their treatment.
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  • Visualizing Tropoelastin in a Long-Term Human Elastic Fibre Cell Culture Model

    Elastin is an essential protein found in a variety of tissues where resilience and flexibility are needed, such as the skin and the heart. When aiming to engineer suitable implants, elastic fibres are needed to allow adequate tissue renewal. However, the visualization of human elastogenesis remains in the dark. To date, the visualization of human tropoelastin (TE) production in a human cell context and its fibre assembly under live cell conditions has not been achieved. Here, we present a long-term cell culture model of human dermal fibroblasts expressing fluorescence-labelled human TE. We employed a lentiviral system to stably overexpress Citrine-labelled TE to build a fluorescent fibre network. Using immunofluorescence, we confirmed the functionality of the Citrine-tagged TE. Furthermore, we visualized the fibre assembly over the course of several days using confocal microscopy. Applying super resolution microscopy, we were able to investigate the inner structure of the elastin–fibrillin-1 fibre network.
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  • BABB Clearing and Imaging for High Resolution Confocal Microscopy: Counting and Sizing Kidney Cells in the 21st Century

    Multipohoton microscopy experiment using Leica TCS SP8 MP and Leica 20x/0.95 NA BABB immersion objective. Understanding kidney microanatomy is key to detecting and identifying early events in kidney disease. Improvements in tissue clearing and imaging have been crucial in this field, and now we report on a novel, time-efficient method to study podocyte depletion in renal glomeruli using a combination of immunofluorescence, optical clearing, confocal microscopy and 3D analysis.
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  • Mammary gland development

    Investigating the Influence of Hormone Signaling Pathways on Mammary Gland Development and the Onset of Breast Cancer

    Over their entire lifetime, 1 out of 8 women can suffer from breast cancer. The risk of developing breast cancer (mammary carcinogenesis) increases with a woman’s age and is related to her reproductive history. The chance of mammary carcinogenesis is less for women who give birth to a child before the age of 30. However, it is now known that the risk of breast cancer also can depend on the number of times a woman experiences elevated blood serum progesterone levels, in relation to the menstrual cycle, before her first pregnancy.
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  • Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

    We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.
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  • How to Prepare Your Specimen for Immunofluorescence Microscopy

    Immunofluorescence (IF) is a powerful method for visualizing intracellular processes, conditions and structures. IF preparations can be analyzed by various microscopy techniques (e.g. CLSM, Epifluorescence, TIRF, GSDIM), depending on the application or the researcher’s interest. Meanwhile, IF has become indispensable for a large number of research groups which have at least access to a simple fluorescence microscope.
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  • Quick Guide to STED Sample Preparation

    This document is intended as a quick reference guide for the most common questions regarding the preparation of samples for the Leica SP8 TCS STED 3X, by briefly explaining the theoretical basics required from samples for stimulated emission depletion. It provides information about the most common immuno-fluorescence labeling techniques, working mounting media, basic quality optimization procedures and experiment designs. It also contains a detailed list of reagents, antibodies and disposables frequently and successfully used in super-resolution STED microscopy. In summary, this guide is meant to present the necessary knowledge, including some tips and tricks, to users that are preparing their first super-resolution microscopy sample.
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  • Sample Preparation for GSDIM Localization Microscopy – Protocols and Tips

    The widefield super-resolution technique GSDIM (Ground State Depletion followed by individual molecule return) is a localization microscopy technique that is capable of resolving details as small as 20 nanometers. GSDIM is suitable for a wide range of samples.
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  • Protein Trafficking in Cereal Seeds

    Seeds accumulate proteins and starch which will be broken down and mobilized upon germination. Among seeds, cereals constitute an example of a highly specialized storage tissue that constitutes up to 80% of the total seed volume (Watson 1987). The seed endomembrane system is highly specialized and seed storage proteins travel through the endomembrane system en route to the protein bodies, which are either derived from the Endoplasmic reticulum (ER) or of vacuolar origin (Muntz 1998).
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  • Intravital Immunofluorescence for Visualizing the Microcirculatory and Immune Microenvironments in the Mouse Ear Dermis

    Intravital imaging of inflammatory and remodeling processes has been an important area of research and has also motivated the creation of numerous transgenic reporter mouse models that express fluorescent proteins. Our paper (Kilarski, Güç et al. 2013) describes a new in vivo imaging technique, which design was based on innovative concept of using immunostaining for live cells and tissue structures on surgically exposed mouse dermis without causing harmful immunotoxic effect.
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  • Fluorescent Dyes

    A basic principle in fluorescence microscopy is the highly specific visualization of cellular components with the help of a fluorescing agent. This can be a fluorescing protein – for example GFP – genetically linked to the protein of interest. If cloning is impossible – for instance in histologic samples – it is required to use other techniques like immunofluorescence staining to visualize the protein of interest.
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  • Subcellular Localization of AKT and Tubulin using Super-Resolution Microscopy

    Stimulated Emission Depletion microscopy, or STED nanoscopy, is a technique that uses the non-linear de-excitation of fluorescent dyes to overcome the resolution limit imposed by diffraction encountered with standard confocal laser scanning microscopes and conventional far-field optical microscopes[1]. Compared to traditional confocal microscopy, STED offers exceptional improvements in resolution allowing visualization of cellular events at unprecedented levels.
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  • Advanced Correlative Light/Electron Microscopy: Current Methods and New Developments Using Tokuyasu Cryosections

    Microscopy is an essential tool for analysis of cellular structures and function. With the advent of new fluorescent probes and super-resolution light microscopy techniques, the study of dynamic processes in living cells has been greatly facilitated. Fluorescence light microscopy provides analytical, quantitative, and three-dimensional (3D) data with emphasis on analysis of live cells using fluorescent markers.
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  • High Data Output and Automated 3D Correlative Light–Electron Microscopy Method

    Correlative light/electron microscopy (CLEM) allows the simultaneous observation of a given subcellular structure by fluorescence light microscopy (FLM) and electron microscopy. The use of this approach is becoming increasingly frequent in cell biology. In this study, we report on a new high data output CLEM method based on the use of cryosections.
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