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Leica VT1200 S Materials Testing

Leica VT1200 S Fully automated vibrating blade microtome

Schematic overview of the experimental design

Preclinical model of organotypic culture for pharmacodynamic profiling of human tumors

Valentina Vaira, Giuseppe Fedele, Saumyadipta Pyne, Ester Fasoli, Giorgia Zadra, Dyane Bailey, Eric Snyder, Alice Faversani, Guido Coggi, Richard Flavin, Silvano Bosari, and Massimo Loda

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

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

Diana M. Mathis1, Jennifer L. Furman2, Christopher M. Norris2, 3

1Graduate Center for Gerontology, University of Kentucky College of Public Health, 2Department of Molecular and Biomedical Pharmacology, University of Kentucky College of Medicine, 3Sanders-Brown Center on Aging, University of Kentucky College of Medicine

 

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.

See the educational video and download the full article as pdf

Leica VT1000 S Microtome with vibrating blade

The localization of Nerve Cell Antigens in rat brain with the Leica VT1000 vibrating blade microtome

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.

Read the complete applications brief by downloading the pdf via the hyperlink.

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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)

Near-monolayer sectioning of live CNS tissue

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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An improved method for preparing thick sections for immuno/histochemistry and confocal microscopy and its use to identify rare events

(P. Monaghan, P.R. Watson, H. Cook, L. Scott, T.S. Wallis, D.Robertson - Journal of Microscopy, August 2001).

Detection of rare events within solid tissues by immunocytochemistry is aided by imaging thick sections. Sections of 40-100 µm thickness of paraformaldehyde-fixed solid tissue can be prepared by use of a vibrating microtome and when immunolabelled these sections can be imaged in a confocal microscope.

This approach provides excellent preservation of the structure of the sample and imposes minimal antigenic damage. In studies of the invasion of the bovine intestinal epithelium by Salmonella, this method has allowed detection of individual invading bacteria within large samples.

The thick vibrating microtome sections were also used for the detection of rare apoptotic cell nuclei identified by TUNEL staining.

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Leica VT1200 S

Patch-clamp recording in brain slices with improved slicer technology

J. R. P. Geiger · J. Bischofberger · I. Vida · U. Fröbe S. Pfitzinger · H. J. Weber · K. Haverkampf · P. Jonas

The use of advanced patch-clamp recording techniques in brain slices, such as simultaneous recording from multiple neurons and recording from dendrites or presynaptic terminals, demands slices of the highest quality.

In this context the mechanics of the tissue slicer are an important factor. Ideally, a tissue slicer should generate large-amplitude and high-frequency movements of the cutting blade in a horizontal axis, with minimal vibrations in the vertical axis.

We developed a vibroslicer that fulfils these in part conflicting requirements. The oscillator is a permanent-magnet-coil-leafspring system. Using an auto-resonant mechano-electrical feedback circuit, large horizontal oscillations (up to 3 mm peak-to-peak) with high frequency (ca. 90 Hz) are generated. To minimize vertical vibrations, an adjustment mechanism was employed that allowed alignment of the cutting edge of the blade with the major axis of the oscillation.

A vibroprobe device was used to monitor vertical vibrations during adjustment. The system is based on the shading of the light path between a lightemitting diode (LED) and a photodiode. Vibroprobe monitoring revealed that the vibroslicer, after appropriate adjustment, generated vertical vibrations of <1 µm, significantly less than many commercial tissue slicers.

Light- and electron-microscopic analysis of surface layers of slices cut with the vibroslicer showed that cellular elements, dendritic processes and presynaptic terminals are well preserved under these conditions, as required for patch-clamp recording from these structures.

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Leica VT1200 vibrating blade microtome

Patch-clamp recording from mossy fiber terminals in hippocampal slices

Josef Bischofberger, Dominique Engel1, Liyi Li1, Joerg RP Geiger2 & Peter Jonas1

1Institute of Physiology, University of Freiburg, Hermann-Herder-Strasse 7, D-79104 Freiburg, Germany. 2Independent Hertie Research Group, Max-Planck Institute for Brain Research, Deutschordenstrasse 46, D-60528 Frankfurt, Germany. Correspondence should be addressed to P.J. (peter.jonas@physiologie.uni-freiburg.de)

Rigorous analysis of synaptic transmission in the central nervous system requires access to presynaptic terminals. However, cortical terminals have been largely inaccessible to presynaptic patch-clamp recording, due to their small size.

Using improved patch-clamp techniques in brain slices, we recorded from mossy fiber terminals in the CA3 region of the hippocampus, which have a diameter of 2–5 µm.

The major steps of improvement were the enhanced visibility provided by high-numerical aperture objectives and infrared illumination, the development of vibratomes with minimal vertical blade vibrations and the use of sucrose-based solutions for storage and cutting.

Based on these improvements, we describe a protocol that allows us to routinely record from hippocampal mossy fiber boutons. Presynaptic recordings can be obtained in slices from both rats and mice.

Presynaptic recordings can be also obtained in slices from transgenic mice in which terminals are labeled with enhanced green fluorescent protein.

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Live cell indicator reveals abundance of living cells throughout slice (P1 rat) (scale bar = 50 µm)

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)

TEST Ultrathin CNS slices: Enhanced visualization of neuron structure for electrophysiology and imaging (Kopie 1)

L. Song, M. Sawchuk, & S. Hochman. Dept. Physiology, Univ. of Manitoba. Winnipeg, MB, Canada R3E 0W3. E-mail: shawn@scrc.umanitoba.ca) Electrophysiological studies using patch clamp recordings in CNS slice preparations involve either ‘blind’ or visual targeting strategies. Recordings from visually-identified neurons generally require specialized upright microscopes equipped with Nomarski optics (DIC) (e.g. Konnerth et al Pflugers Arch.

414:600, 1989). Further image optimization to observe neuronal processes employ video-enhanced infrared wavelength illumination (e.g. Stuart et al Pflugers Arch. 423:511, 1993). Though powerful, these approaches require specialized, expensive equipment. Image: Live (green) and dead (red) cell assay showing abundance of living cells throughout slice in a P8 rat (scale bar = 100µm)

Since the ability to resolve detailed cellular features is generally limited to 40-50 mm from the slice surface, we have developed an alternate strategy to visualize neurons using a semi-transparent ‘ultrathin’ slice preparation (20-50 mm) visualized on an inverted microscope equipped with Hoffman modulation optics.

Isolated cerebellum, hippocampus or spinal cord from neonatal rats (P1- P14) were embedded in AGAR (2.5% w/v) then sectioned with a Leica VT1000E vibrating blade microtome. After incubation at 32°C, slices were fixed to the bottom of the recording chamber and maintained at room temperature.

The enhanced transparency due to reduced slice thickness permitted superior optical resolution of dendritic and axonal processes and cells were visible throughout the slice thickness. Image: Live cell indicator reveals abundance of living cells throughout slice (P1 rat) (scale bar = 50 µm)

ve/dead cell staining revealed that many cells remained viable for imaging and electrophysiological experimentation. In all CNS regions examined, neurons were easily identified for successful patch recordings.

While not yet tested, use of an inverted microscope should permit high numerical aperture oil-immersion objectives to be used for simultaneous imaging experiments and the reduced slice thickness would hasten drug equilibration and washout times. Supported by the Canadian Neuroscience Network.

Image: 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)

<media 21153 - download>Hier haben wir einen PDF-Link</media>

Live cell indicator reveals abundance of living cells throughout slice (P1 rat) (scale bar = 50 µm)

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)

Ultrathin CNS slices: Enhanced visualization of neuron structure for electrophysiology and imaging

L. Song, M. Sawchuk, & S. Hochman. Dept. Physiology, Univ. of Manitoba. Winnipeg, MB, Canada R3E 0W3. E-mail: shawn@scrc.umanitoba.ca) Electrophysiological studies using patch clamp recordings in CNS slice preparations involve either ‘blind’ or visual targeting strategies. Recordings from visually-identified neurons generally require specialized upright microscopes equipped with Nomarski optics (DIC) (e.g. Konnerth et al Pflugers Arch.

414:600, 1989). Further image optimization to observe neuronal processes employ video-enhanced infrared wavelength illumination (e.g. Stuart et al Pflugers Arch. 423:511, 1993). Though powerful, these approaches require specialized, expensive equipment. Image: Live (green) and dead (red) cell assay showing abundance of living cells throughout slice in a P8 rat (scale bar = 100µm)

Since the ability to resolve detailed cellular features is generally limited to 40-50 mm from the slice surface, we have developed an alternate strategy to visualize neurons using a semi-transparent ‘ultrathin’ slice preparation (20-50 mm) visualized on an inverted microscope equipped with Hoffman modulation optics.

Isolated cerebellum, hippocampus or spinal cord from neonatal rats (P1- P14) were embedded in AGAR (2.5% w/v) then sectioned with a Leica VT1000E vibrating blade microtome. After incubation at 32°C, slices were fixed to the bottom of the recording chamber and maintained at room temperature.

The enhanced transparency due to reduced slice thickness permitted superior optical resolution of dendritic and axonal processes and cells were visible throughout the slice thickness. Image: Live cell indicator reveals abundance of living cells throughout slice (P1 rat) (scale bar = 50 µm)

ve/dead cell staining revealed that many cells remained viable for imaging and electrophysiological experimentation. In all CNS regions examined, neurons were easily identified for successful patch recordings.

While not yet tested, use of an inverted microscope should permit high numerical aperture oil-immersion objectives to be used for simultaneous imaging experiments and the reduced slice thickness would hasten drug equilibration and washout times. Supported by the Canadian Neuroscience Network.

Image: 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)