Applications Leica VT1200

(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

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

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)

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)