Leica CM3050 S Research Cryostat
Knife Angle in Microtomy
Tips & Tricks in Sample Preparation
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.
Click on the hyperlink above to read the entire article.
One Pathologist’s Approach to Teaching Frozen Sectioning
Anyone who has visited Dr. Peter’s website tutorial on frozen section technique knows that he is a true believer in the brush technique. Let’s learn how he teaches this valuable skill to his residents:
The first step is to find a comfortable hand position to maximize fine motor ability for this very delicate maneuver. For this, I have my students hold the brush like a pen and gently rest a finger on the anti-roll plate or at the edge of the cryostat stage.
Next, I tell them to try to write their names on the edge of the block with the brush to gain a sense of the fine movements they will use in the sectioning process.
To learn more about this pathologist’s approach to cryosectioning visit Dr. Peter’s tutorial at: http://pathologyinnovations.com/frozen_section_technique.htm.
Drug Localization in Tissues and Cells
Receptor Micro Autoradiography
Receptor Micro-Autoradiography was developed for the tissue localization of drugs. Since drugs are generally small molecular weight compounds acting through weak binding to specific receptors with limited capacity, the method is designed to provide high resolution and high sensitivity, and at the same time to preserve microscopic tissue structure with the compound retained at its original site in vivo. This method has been tested before its introduction with compounds known localized and applied for several decades to the study of drugs, demonstrating its high diagnostic and predictive value.
Receptor micro-autoradiography is based on the use of radiolabeled (predominantly 3H or 125I) compounds with high specific activity in conjunction with nuclear emulsion-coated slides and thin frozen sections cut in a cryostat.
Cellular and subcellular detail can be viewed. Associated radioactivity can be characterized and quantified. Time- and dose-related data of different compounds can be compared. A receptor-drug-homunculus can be created as a computer model to provide overviews and details about sites of deposition and specific binding, receptor-drug half lives, hierarchies of target affinities, and indications for therapeutic indices, side effects and toxicity.
Documentation of precise localization can give important clues for drug functions and can serve as a guide for histochemical, biochemical, functional and clinical follow-up for the development of new therapeutic applications. With its high diagnostic and predictive qualities, receptor micro-autoradiography is an indispensable tool for drug research and development.
Detailed information and instructions on the most effective experimental design, execution of technique, and evaluation of autoradiograms are contained in a recently published manual. The publication is called "Drug Localization in Tissues and Cells - Receptor Microscopic Autoradiography. A Basis for Tissue and Cellular Pharmacokinetics, Drug Targeting, Delivery, and Prediction", author: Walter E. Stumpf .
The book is available through the:
University of North Carolina
Bookstore in Chapel Hill (UNC Student Stores, Chapel Hill, NC 27599).
Please direct order inquiries to:
Phone: (919) 962-2420
Fax: (919) 962-9661
The Art of Embedding Tissue for Frozen Section
(by Stephen R. Peters, Dept. of Pathology, Hackensack University Medical Center, New Jersey, USA)
This article describes a system used for embedding of tissues for the preparation of frozen sections.
This novel system uses simple techniques and apparatus to accomplish face-down embedding in freezing-temperature steel wells.
The system is easy to learn and offers many advantages over conventional methods, including speed, high precision and predictability, and reduced tissue wastage.
The system is easily adapted in most existing cryostats, used no additional consumables, and requires minimal maintenance. (The J Histotechnol 26:11, 2003)
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New approach to preparing thin whole-body sections for histochemistry, immunohistochemistry, and light microscopical autoradiography.
(Tadafumi Kawamoto, Radioactive-Isotope Research Laboratories, Tsurumi University, School of Dental Medicine, 2-1-3 Tsurumi, Tsurumi-Ku, Yokohama, Japan)
This study presents a method for preparing fresh-frozen sections that can be used for histological, histochemical and immunohistochemical studies at light microscopical level with entire laboratory animals.
To achieve this, an adhesive film to support the frozen cut section was specially prepared. In addition, the cryomicrotome (CM 3500) and cryostat were modified. An entire child rat and a pregnant mouse were frozen in cooled hexane (-94°C).
The frozen animal was embedded in a 5% CMC gel. The animal and gel were completely frozen. The frozen CMC block was trimmed with a disposable tungsten carbide blade at approximately –23°C.
The exposed tissue surface was covered with an adhesive film and cut into 2-5 micro-meter thick sections.