Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving fixed or stabilized – nonliving microorganism,...
Reexamination Certificate
2001-09-10
2003-04-29
Gitomer, Ralph (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving fixed or stabilized, nonliving microorganism,...
C435S040520
Reexamination Certificate
active
06555334
ABSTRACT:
FIELD OF INVENTION
This invention relates to the field of histology and immuno-histology using immunoelectron microscopy. More specifically, this invention relates to the field of free-floating cryostat sections for use in light and electron microscopy to bridge the gap between these two viewing mediums.
BACKGROUND OF THE INVENTION
In many instances, a scientist studying a histological sample desires to view a tissue specimen with both a light microscope and also with an electron microscope to obtain information that is unique to each discipline. Unfortunately, there is no known way to view the exact same tissue sample with both devices. No medium has been developed that allows the scientist to move a single tissue specimen from one device to the other.
In the past, two separate tissue samples from the same subject were required for viewing with the separate devices. A comparison had to be made to bridge the gap between the results of the light microscope and the electron microscope. However, the comparison of two separate samples has inherent limitations.
Processing of tissues for electron microscopy routinely required that tissues be chemically fixed. Some of the practices that have been used are thick paraffin embedded tissue sectioning, and microwave processing. These techniques require chemical fixation with formaldehyde or combinations of aldehydes. In regard to paraffin processing, thick sections subsequently processed for electron microscopy are structurally compromised as a result of the necessary exposure to highly volatile reagents required for adequate paraffin processing (zylenes, toluenes). In the case of microwave energy, the tissues are typically subjected to a dilute concentration of chemical fixatives for a short time (seconds) prior to exposing the tissue to microwave processing. The effect of the microwave is to accelerate the chemical fixation process by superheating and/or by producing rapidly oscillating water molecules within the tissue. Both effects of heat and water oscillations act to etch or open the tissue to the dilute fixative.
However, in studies that address the preservation of labile structural proteins, chemical fixation is contraindicated. Therefore other means of preservation were pursued. Procedures presently used that circumvent chemical fixation are cryofixation, microwave-assisted fixation and vibratoming.
Cryofixed tissues for electron microscopy require that the tissues then be cryosectioned through the use of cryoultramicrotomes. These procedures (cryofixation and cryoultramicrotomy) are terribly expensive (cryoultratomes) and extremely labor intensive requiring committed technical dedication by a well-trained technical staff. Moreover, light microscopic examinations and electron microscopy examinations within the same study require the use of separate and distinct tissue sections for each discipline.
Microwave assisted fixation uses chemical fixation that is accelerated by action of the microwave.
Vibratomed fresh (unfixed tissues) for subsequent processing is yet another method that has been employed. “Vibratoming” makes use of a rapidly oscillating microtome blade which sections through unfixed tissue. However only certain tissues can be vibratomed. The types of unfixed tissues that can be vibratomed successfully are terribly restricted with skin being the least desirable.
In these currently existing practices, there are the following limitations: 1. All except vibratoming require some form of chemical fixation to retain the structural integrity of the tissue sample especially at the ultrastructural level (electron microscopy); 2. All practices that require chemical fixation preclude the preservation of some important antigenic determinants of skin basement membrane proteins; 3. Loss of antigenic determinants then precludes the use of special staining practices (immunochemistry) which would visualize these proteins at light and electron microscopy levels.
As stated, there are particular types of tissues that are especially difficult to work with for purposes of microscope viewing. One such tissue type is skin basement membrane zone. Since skin basement membrane protein components are labile to conventional chemical fixation and since skin is not amenable to vibratome sectioning, frozen skin sections are routinely used for light microscopic immunohistochemical study of the skin basement membrane zone. This requires the use of conventional frozen sections. However, inherent limitations of conventional frozen sections, including compromised morphology and requirement for glass slide-mounting, usually limit study solely to the light microscopic level. These same sections cannot subsequently be used for observation by an electron microscope.
Sections of skin basement membrane zone that are mounted on slides for use with light microscopes are routinely discarded after they are viewed under the light microscope. New and separate sections from the same animal specimen must be taken for preparation and viewing by an electron microscope. This process is both expensive, time consuming and requires comparison of two separate and distinct tissue specimens. A single specimen cannot be subsequently used for viewing with the electron microscope.
Immunoelectron microscopy remains a method of choice for determining the precise immunoanatomical location of antigens at the ultrastructural level (Schaumburg-Lever, 1995; 1999). This technique has been used extensively for mapping the distribution of epidermal-dermal junction proteins in various inherited bullous diseases including bullous pemphigoid (Bedane et al., 1997) and dystrophic epidermolysis bullosa (Bruckner-Tuderman et al., 1989). Recently, immunoelectron microscopy was employed to study basement membrane proteins in sulfur mustard-induced cutaneous vesicating lesions (Monteiro-Riviere and Inman, 1995; Petrali and Oglesby-Megee, 1997). A persistent concern of immunoelectron microscopy is the preservation of ultrastructural morphology while simultaneously maintaining the immunoreactivity of proteins of interest. This concern becomes a special problem when antigenic epitopes of proteins are vulnerable to damage by chemical fixation rendering antigenic binding sites insensitive to antibody labeling.
The present invention advances the use of immunoelectron microscopy to study the immunopathology of the skin basement zone induced by the chemical vesicating agent sulfur mustard. Since proteins of the skin basement membrane zone are susceptible to chemical fixation-induced damage, immunoidentification of basement membrane proteins is restricted largely to unfixed cryostat sections mounted onto microscope slides. Further evaluation of these sections by electron microscopy requires that tissue be separated from slides by the “pop-off” method introduced by Bretschneider and colleagues (1981). This useful, but arduous technique causes loss of valuable material when tissue sections do not separate from the slide cleanly and often yields poor ultrastructural detail (Bretschneider et al., 1981). To circumvent these technical problems, the present invention employs a procedure using unfixed, free-floating frozen skin sections for immuno-detection of cutaneous basement membrane zone components of interest in the study of vesicating skin lesions. The adaptation of a free-floating method to bridge the light microscope and electron microscope gap in the area of basement membrane biology is novel and can be readily applied to all areas of basement membrane research.
The present invention is especially advantageous because it avoids chemical fixation by using cryofixation which promotes the stability of labile proteins, performs procedures to identify antigenicity (viability) of proteins and their anatomical locations (immunohistochemistry), performs these procedures on the same tissue section, processes the same immunostained tissue section for both light and electron microscopic analysis, and performs these procedures on skin tissue that is not amenable to vibratome sectioning.
In addition to the above, the ap
Hamilton Tracey A.
Kan Robert Kwai
Oglesby-Megee Susan B.
Petrali John P.
Arwine Elizabeth
Gitomer Ralph
The United States of America as represented by the Secretary of
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