Springer protocols feed by Molecular Medicine 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Whole-Mount Skeletal Staining
The first step in almost every investigation of skeletal phenotypes is analysis of whole-mount skeletal preparations. Whole-mount skeletal staining permits evaluation of the shapes and sizes of skeletal elements in their appropriate locations. The technique is thus the major method for detecting changes in skeletal patterning. Because cartilage and bone can be distinguished by differential staining, this technique is also a powerful means to assess the pace of skeletal maturation. This protocol covers staining of the pre- and postnatal mouse skeleton using Alcian blue and Alizarin red to identify cartilage and bone, respec...
Source: Springer protocols feed by Molecular Medicine - February 3, 2014 Category: Molecular Biology Source Type: news
TUNEL Assay on Skeletal Tissue Sections to Detect Cell Death
At least two distinct modalities of cell death, apoptosis and necrosis, can be distinguished based on differences in morphological, biochemical, and molecular changes of dying cells [1, 2]. Cell death is involved both in physiological and pathological conditions of the skeleton: for example, apoptosis is a crucial event during limb development. Therefore, detection of cell death by using a simple stain is a powerful tool to study molecular and cellular mechanisms of skeletal development and repair [3]. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - February 3, 2014 Category: Molecular Biology Source Type: news
Proliferation Assays (BrdU and EdU) on Skeletal Tissue Sections
Assessing cell proliferation in situ is an important phenotyping component of skeletal tissues from development to adult stages and disease. Various methods exist including immunostaining for proteins and protein modifications associated with specific steps of the cell cycle, but the gold standard is to quantify the percentage of DNA-synthesizing cells. The thymidine analog 5-bromo-2′-deoxyuridine (BrdU) has been widely used in the last decades for this purpose, with the inconvenience that its detection is lengthy and requires harsh treatment of tissue sections to give access of anti-BrdU antibody to nucleosides in g...
Source: Springer protocols feed by Molecular Medicine - February 3, 2014 Category: Molecular Biology Source Type: news
Radioactive In Situ Hybridization to Detect Gene Expression in Skeletal Tissue Sections
In situ hybridization (ISH) using RNA probes is a valuable technique to characterize gene expression patterns in animal tissues. It provides valuable spatial information about gene expression. Compared to the nonradioactive alternatives,35S radioactive ISH generally provides higher sensitivity. Here, we describe the procedure for35S ISH on paraffin sections from the skeletal tissues of mouse embryos. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - February 3, 2014 Category: Molecular Biology Source Type: news
Nonradioactive In Situ Hybridization on Skeletal Tissue Sections
Spatial and temporal visualization of RNA transcripts in tissue is a key tool in studying both developmental and pathological processes. In situ hybridization is a highly sensitive method for RNA transcript detection. It is based on sequence complementation between a labeled RNA probe and the RNA transcript of interest. The labeled probe is then detected by immunohistochemical methods using an antibody conjugated to an enzyme that catalyzes the generation of chromogenic or fluorescent signals, which indicate the location of the transcript. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - February 3, 2014 Category: Molecular Biology Source Type: news
Whole-Mount In Situ Hybridization on Murine Skeletogenic Tissues
Whole-mount in situ hybridization (WISH) is a highly sensitive method for spatial and temporal visualization of RNA transcripts throughout an entire tissue. This method is an excellent tool for studying both embryonic development and disease. It is based on sequence complementation between a Digoxigenin (DIG)-labeled antisense RNA probe and the sense RNA transcript of interest in the tissue. The labeled probe is then visualized by immunohistochemical methods using an antibody conjugated to alkaline phosphatase that catalyzes a color reaction. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - February 3, 2014 Category: Molecular Biology Source Type: news
Beta-Galactosidase Staining in the Skeleton
The lacZ gene, encoding for the β-galactosidase enzyme, is widely used as a reporter gene in bone biology due to the ease of visualization in situ on whole-mount or on tissue sections. In this protocol we provide detailed methods for visualizing this reporter gene for both in vivo and in vitro studies. In addition, we describe a few tips for troubleshooting for mineralized tissues such as the skeleton. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - February 3, 2014 Category: Molecular Biology Source Type: news
Demineralized Murine Skeletal Histology
Cartilage and bone are specialized skeletal tissues composed of unique extracellular matrices. Bone, in particular, has a highly calcified or mineralized matrix that makes microtomy and standard histological studies very challenging. Therefore, methods to appropriately fix and decalcify mineralized skeletal tissues have been developed to allow for paraffin processing and standard microtomy. In this chapter, we illustrate methods for tissue grossing, fixation, decalcification, paraffin processing, embedding, sectioning, and routine histological staining of demineralized murine skeletal tissues. We also discuss methods for d...
Source: Springer protocols feed by Molecular Medicine - February 3, 2014 Category: Molecular Biology Source Type: news
Preparation of Thin Frozen Sections from Nonfixed and Undecalcified Hard Tissues Using Kawamot’s Film Method (212)
A method for preparing hard tissue sections by using an adhesive film is described. The method produces very thin (to one-micrometer thick) frozen sections from adult mouse and rat bone. The bone tissue is freeze-embedded with water-soluble medium and then cut with a disposable tungsten carbide blade after mounting the adhesive film onto the cut surface. The sections are stained on the adhesive film and preserved between the adhesive film and glass slide. All the steps including the embedding, cutting, staining, and mounting are completed within only 20 min. The soft and hard tissues are preserved satisfactorily and the bo...
Source: Springer protocols feed by Molecular Medicine - February 3, 2014 Category: Molecular Biology Source Type: news
Tips and Techniques for Processing and Sectioning Undecalcified Murine Bone Specimens
Preparation of mineralized tissue specimens for bone-specific staining encompasses a critical sequence of histological techniques that provides visualization of tissue and cellular morphology. Bone specimens are fixed in 10 % neutral-buffered formalin, dehydrated in graded ethanol (EtOH) solutions (and optionally cleared in xylene), infiltrated and embedded in polymethyl methacrylate (methyl methacrylate), classically sliced into 4–10 micrometer (μm) sections, and stained with bone-specific histological stains such as von Kossa (with either nuclear fast red solution counterstain or MacNeal’s tetrachrome coun...
Source: Springer protocols feed by Molecular Medicine - February 3, 2014 Category: Molecular Biology Source Type: news
Renal Capsule Transplantations to Assay Skeletal Angiogenesis
Renal capsule transplantation is a very helpful method to grow embryonic tissues or tumors in a vascular environment, allowing long-term engraftment and biological analyses. This chapter describes the surgical procedure for the transplantation of embryonic skeletal elements in the renal capsule of adult mice and points out the manipulations that can be applied for assaying the role of angiogenesis during bone development. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - February 3, 2014 Category: Molecular Biology Source Type: news
Cartilage Explant Cultures
To investigate chondrocyte biology in an organized structure, limb explant cultures have been established that allow the cultivation of the entire cartilaginous skeletal elements. In these organ cultures, the arrangement of chondrocytes in the cartilage elements and their interaction with the surrounding perichondrium and joint tissue are maintained. Chondrocyte proliferation and differentiation can thus be studied under nearly in vivo conditions. Growth factors and other soluble agents can be administered to the explants, and their effect on limb morphogenesis, gene expression, and cell–matrix interactions can be st...
Source: Springer protocols feed by Molecular Medicine - February 3, 2014 Category: Molecular Biology Source Type: news
A Mouse Model of Flexor Tendon Repair
Mouse models offer invaluable cellular and molecular tools for the study of human pathologies including those associated with fibrotic and musculoskeletal diseases. In this methods manuscript, we describe a mouse model of repair and segmental reconstruction of flexor tendons, which in our laboratory has been an invaluable model to study tendon scarring and adhesions. Specifically, we describe in details all the surgical procedures involved, as well as the associated endpoint biomechanical assessments including a novel test of the flexion of the metatarsophalangeal joint as a measure of adhesions, and a standard protocol fo...
Source: Springer protocols feed by Molecular Medicine - February 3, 2014 Category: Molecular Biology Source Type: news
Surgical Induction of Posttraumatic Osteoarthritis in the Mouse
Given the prevalence and the scope of the personal and societal burden of OA, investigators have become increasingly interested in understanding the pathogenic basis of disease and developing novel disease-modifying OA therapies. Because of the well-documented central role that joint trauma plays in the initiation of knee OA, large animal and rodent models of knee injury that accurately recapitulate the OA disease process have become increasingly widespread over the past decade. To enable study in the context of defined genetic backgrounds, investigative teams have informally developed standardized protocols for injuring t...
Source: Springer protocols feed by Molecular Medicine - February 3, 2014 Category: Molecular Biology Source Type: news
The Murine Femoral Bone Graft Model and a Semiautomated Histomorphometric Analysis Tool
Preclinical studies of bone repair remain a high priority because of unresolved clinical problems associated with treating critical segmental defects and complications of fracture healing. Over the last decade, the murine femoral allograft model has gained popularity due to its standardized surgery and potential for examining a vast array of radiographic, biomechanical, and histological outcome measures. Here, we describe these methods and a novel semiautomated histomorphometric approach to quantify the amount of bone, cartilage, and undifferentiated mesenchymal tissue in demineralized paraffin sections of allografted muri...
Source: Springer protocols feed by Molecular Medicine - February 3, 2014 Category: Molecular Biology Source Type: news
