Reviewer
Emilie Viennois
  • Assistant Professor, INSERM
Research fields
  • Cancer Biology, Microbiology, Molecular Biology
A Fast and Efficient Decellularization Method for Tissue Slices

The study and use of decellularized extracellular matrix (dECM) in tissue engineering, regenerative medicine, and pathophysiology have become more prevalent in recent years. To obtain dECM, numerous decellularization procedures have been developed for the entire organ or tissue blocks, employing either perfusion of decellularizing agents through the tissue’s vessels or submersion of large sections in decellularizing solutions. However, none of these protocols are suitable for thin tissue slices (less than 100 µm) or allow side-by-side analysis of native and dECM consecutive tissue slices. Here, we present a detailed protocol to decellularize tissue sections while maintaining the sample attached to a glass slide. This protocol consists of consecutive washes and incubations of simple decellularizing agents: ultrapure water, sodium deoxycholate (SD) 2%, and deoxyribonuclease I solution 0.3 mg/mL (DNase I). This novel method has been optimized for a faster decellularization time (2–3 h) and a better correlation between dECM properties and native tissue-specific biomarkers, and has been tested in different types of tissues and species, obtaining similar results. Furthermore, this method can be used for scarce and valuable samples such as clinical biopsies.

Parasitemia Evaluation in Mice Infected with Schistosoma mansoni
Authors:  Naiara Clemente Tavares and Marina Moraes Mourão, date: 05/20/2021, view: 3246, Q&A: 0

Schistosomiasis is a neglected tropical disease. Its treatment relies on the use of a single drug, praziquantel. Due to treatment limitations, an alternative for schistosomiasis chemotherapy is required; thus, a better understanding of parasite biology and host-parasite interactions is valuable to aid the identification of new anti-Schistosoma drugs. The parasite has a complex life cycle, which results in challenges regarding the evaluation of Schistosoma mansoni development and mammalian infection establishment. Accordingly, this protocol describes methodologies to evaluate: (1) adult worm growth; (2) reproduction; and (3) granuloma formation; and consequently allows more comprehensive knowledge of S. mansoni development in a natural biological system.

Attachment of a 32P-phosphate to the 3′ Terminus of a DNA Oligonucleotide
Authors:  Joshua C. Cofsky and Jennifer A. Doudna, date: 10/20/2020, view: 3727, Q&A: 0
Biochemical investigations into DNA-binding and DNA-cutting proteins often benefit from the specific attachment of a radioactive label to one of the two DNA termini. In many cases, it is essential to perform two versions of the same experiment: one with the 5′ DNA end labeled and one with the 3′ DNA end labeled. While homogeneous 5′-radiolabeling can be accomplished using a single kinase-catalyzed phosphorylation step, existing procedures for 3′-radiolabeling often result in probe heterogeneity, prohibiting precise DNA fragment identification in downstream experiments. We present here a new protocol to efficiently attach a 32P-phosphate to the 3′ end of a DNA oligonucleotide of arbitrary sequence, relying on inexpensive DNA oligonucleotide modifications (2′-O-methylribonucleotide and ribonucleotide sugar substitutions), two enzymes (T4 polynucleotide kinase and T4 RNA ligase 2), and the differential susceptibility of DNA and RNA to hydroxide treatment. Radioactive probe molecules produced by this protocol are homogeneous and oxidant-compatible, and they can be used for precise cleavage-site mapping in the context of both DNase enzyme characterization and DNA footprinting assays.

Graphic abstract


Glioma Induction by Intracerebral Retrovirus Injection
Authors:  Ravinder K Verma , Fanghui Lu and Qing Richard Lu, date: 07/20/2017, view: 9369, Q&A: 0
Glioblastoma (GBM) is the most common primary brain cancer in adults and has a poor prognosis. It is characterized by a high degree of cellular infiltration that leads to tumor recurrence, atypical hyperplasia, necrosis, and angiogenesis. Despite aggressive treatment modalities, current therapies are ineffective for GBM. Mouse GBM models not only provide a better understanding in the mechanisms of gliomagenesis, but also facilitate the drug discovery for treating this deadly cancer. A retroviral vector system that expresses PDGFBB (Platelet-derived growth factor BB) and inactivates PTEN (Phosphatase and tensin homolog) and P53 tumor suppressors provides a rapid and efficient induction of glioma in mice with full penetrance. In this protocol, we describe a simple and practical method for inducing GBM formation by retrovirus injection in the murine brain. This system gives a spatial and temporal control over the induction of glioma and allows the assessment of therapeutic effects with a bioluminescent reporter.
Spore Preparation Protocol for Enrichment of Clostridia from Murine Intestine
Authors:  Eric M. Velazquez, Fabian Rivera-Chávez and Andreas J. Bäumler, date: 05/20/2017, view: 7200, Q&A: 0
In recent years, many spore-forming commensal Clostridia found in the gut have been discovered to promote host physiology, immune development, and protection against infections. We provide a detailed protocol for rapid enrichment of spore-forming bacteria from murine intestine. Briefly, contents from the intestinal cecum are collected aerobically, diluted and finally treated with chloroform to enrich for Clostridia spores.
Isolation of Outer Membrane Vesicles from Phytopathogenic Xanthomonas campestris pv. campestris
Authors:  Gideon Mordukhovich and Ofir Bahar, date: 03/05/2017, view: 11442, Q&A: 5
Gram-negative bacteria naturally release outer membrane vesicles (OMVs) to the surrounding environment. OMVs contribute to multiple processes, such as cell-cell communication, delivery of enzymes and toxins, resistance to environmental stresses and pathogenesis. Little is known about OMVs produced by plant-pathogenic bacteria, and their interactions with host plants. The protocol described below discusses the isolation process of OMVs from Xanthomonas campestris pv. campestris strain 33913, a bacterial pathogen of Crucifiers. Nevertheless, this protocol can be used and/or adapted for isolation of OMVs from other phytopathogenic bacteria to promote the study of OMVs in the context of plant-microbe interactions.
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