Applications Leica VT1200

Schematic overview of the experimental design

Abstract

Predicting drug response in cancer patients remains a major challenge in the clinic. We have perfected an ex vivo, reproducible, rapid and personalized culture method to investigate antitumoral pharmacological properties that preserves the original cancer microenvironment. Response to signal transduction inhibitors in cancer is determined not only by properties of the drug target but also by mutations in other signaling molecules and the tumor microenvironment. As a proof of concept, we, therefore, focused on the PI3K/Akt signaling pathway, because it plays a prominent role in cancer and its activity is affected by epithelial–stromal interactions. Our results show that this culture model preserves tissue 3D architecture, cell viability, pathway activity, and global gene-expression profiles up to 5 days ex vivo. In addition, we show pathway modulation in tumor cells resulting from pharmacologic intervention in ex vivo culture. This technology may have a significant impact on patient selection for clinical trials and in predicting response to small-molecule inhibitor therapy. Click to read the entire report.

Leica VT1000 P

Abstract

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid.

Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)^1-3.

Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems.

Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

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The knife angle is important to the application

Charles W. Scouten, Ph.D., Leica Microsystems

To prepare biological tissue for observation under a microscope, the tissue is usually cut in thin slices. Most biological tissue is too soft to cut; the knife would push into it and compress it, even if the cutting edge was very sharp.

Therefore, the tissue is either frozen and sectioned in a cryostat or embedded in a hardening material like paraffin or resin, or cut while still soft with a vibrating blade microtome. The correct knife angle is the subject of much misunderstanding, misleading experience, and incorrect information passed between microtomists, but in fact can be logically derived.

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Leica VT1000 S Microtome with vibrating blade

Immunohistochemistry is an important research method to study the central nervous system (CNS). During the last three decades, different immunodetection systems have been developed.

CNS antigens can be detected by isotopic, enzymatic and fluorescence systems. Although these methods can be used on brain sections obtained with a cryostat, more accurate localization and superior morphological detail requires the use of non-frozen tissue.

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Immunostaining of CAA1 glial cells with GFAP (scale bar = 50 µm) (for slice culture procedures, see Parsley, C.P. et al. Society for Neurosci. Abstr. 230.5, 1996)

The in-situ study of sections of living tissue maintained in-vitro is a powerful method to elucidate many aspects of cellular and network function in the CNS, both in acute slice and long-term culture (organotypic) preparations.

Recently, there has been an increased demand for technologies that enhance optical resolution at the synaptic/cellular level in order to better identify particular cell types or examine the properties of distinct spatial regions within individual cells.

Applications brief from Shawn Hochman et al (Univ. of Manitoba, Canada) and Claudia Dorenkamp (Leica Biosystems Nussloch GmbH)

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