Applications Leica VT1000 S

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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(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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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)