Bleaching and dyeing; fluid treatment and chemical modification – Dyeing process utilizing electric – magnetic – or wave energy;...
Reexamination Certificate
1999-06-11
2002-06-25
Warden, Jill (Department: 1743)
Bleaching and dyeing; fluid treatment and chemical modification
Dyeing process utilizing electric, magnetic, or wave energy;...
C204S450000, C204S600000
Reexamination Certificate
active
06409774
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to the staining of biological samples for imaging.
The preparation of organic tissue samples and other materials for transmission microscopy, both visible light and electron microscopy, is normally carried out by subjecting the sample to a series of chemical treatments culminating in the production of a solid block in which the sample is embedded.
In a conventional tissue preparation process, the tissue is first chemically fixed with formalin, glutaraldehyde, or other material which serves to preserve the sample from autolysis (self-degradation), to render the sample rigid, and to increase its permeability, thereby enhancing the infiltration of the subsequent solutions. The infiltration steps which follow chemical fixing remove all of the water from the sample through progressive replacement of water with increasing concentrations of solvents such as alcohol and xylene. Infiltration is followed by treatment with melted paraffin and the sample then is cooled to room temperature whereupon it solidifies. Alternatively, the tissue is infiltrated with plastic polymer that is then hardened by heat, ultraviolet light or other means. The hardened, infiltrated tissue is then position in a mold and surrounded with paraffin or plastic to produce a tissue block.
The block containing the tissue is sectioned on a microtome. In the case of light microscopy, the sections are collected and placed on glass slides. Once secured on the slides, the sections are then stained with any number of dyes which label particular parts of the cell (e.g., nucleic acids, lipids, etc.) or, altematively, are processed for immunohistochemistry.
Alternatively, methods are available for en bloc staining, wherein the entire sample is stained by immersion, prior to being subjected to infiltration and embedment to produce the tissue block. Sections are then cut from the block for transmission microscopy, or the cut face of the block itself is imaged in a process called block face microscopy. In the latter case, including that implemented in the Surface Imaging Microscope (U.S. Pat. No. 4,960,330, “Image Recording Apparatus”), a sample that has been stained en bloc is subsequently infiltrated by and embedded in a medium that is heavily opacified or otherwise treated to allow for the suppression of images of tissue originating from more than a few microns deep in the block. This results in the production of a thin, “virtual section” closely resembling a conventional glass-slide mounted tissue section.
Because the volume of material imaged in a block face microscopy is large relative to the much thinner section of material used in conventional glass slide-based technology, en bloc staining must accomplish penetration through a greater thickness of material. This circumstance results in an undesirable increase in sample preparation time.
Methods employed to accelerate the penetration of stain into the tissue in en bloc staining include the proper choice of fixative; the application of heat, microwave radiation, vacuum or ultrasound; and the addition of detergents and other chemicals to the staining solutions. However, even with the use of such methods, the penetration of stains into relatively large volumes of tissue can require times measured in days or even weeks. Penetration times become even greater when the stain molecule or stain precursor is conjugated with large carrier molecules, such as are used in the practice of immunohistochemistry.
SUMMARY OF THE INVENTION
In general, the invention features a means for increasing the rate and extent of penetration of a component used in staining into a biological tissue by use of electrophoresis. Electrophoresis refers to a process in which charged molecules are caused to migrate in a fluid under the influence of an electric field. The stain or components used in staining include free dyes or colorant reagent molecules, chemical conjugates with antibodies, nucleic acid probes and other high molecular weight carrier molecules, e.g. antibodies.
The invention features a method for en bloc staining a biological sample, including the steps of (a) immersing the sample in a staining solution including an ionically conductive solution and a charged stain molecule or stain precursor that associates with a component of the sample; and (b) applying an electric field across the staining solution, whereby the stain molecule or precursor migrates through the sample. Penetration of the stain into the sample is enhanced.
Another feature of the method of the invention includes (c) immersing the primary antibody-containing sample in a staining solution including an ionically conductive solution and a charged secondary antibody that binds to the primary antibody; and (d) applying an electric field across the staining solution. Penetration of the secondary antibody into the tissue is enhanced.
In another embodiment of the invention, the primary antibody-containing sample is immersed in a solution comprising a secondary antibody that binds to the primary antibody.
In one embodiment of the invention, the sample is infiltrated with a gel support prior to immersing the sample in the staining solution.
In preferred embodiments, a voltage in the range of 50-700V, and preferably 100-400 V, is applied. The electric field is maintained for a time in the range of 1 to 25 hours, and preferably, 3 to 15 hours.
In other preferred embodiments, the staining solution comprises both a stain precursor and a stain molecule, or primary and secondary antibodies, where the pair of molecules is selected to migrate in opposite directions in an electric field. One of the components, for example, a stain precursor, is introduced into the staining solution on a first side of the sample and the second component, for example, a stain molecule, is introduced into the staining solution on the opposing side of the sample.
In another aspect of the invention, the proportion of unbound stain molecule in a sectioned tissue is reduced by (a) first immersing the tissue sample to in a staining solution including an tonically conductive solution and a stain precursor that associates with a component of the sample; and (b) then applying an electric field across the staining solution, whereby penetration of the stain precursor into the sample and emigration of unbound stain precursor from the sample occurs.
The term “sample” is used herein to refer to the tissue or other material which is to be stained and embedded. The sample, prior to staining, has not been prepared by microtomy. The thickness of a sample typically is greater than 200 microns, and can be greater than one millimeter, one centimeter, or more.
The terms “stain” or “stain molecule” are used herein to refer to a dye, reagent, or other material for producing coloration in tissues or microorganisms for microscopic examination. The stain may be a free dye or colorant reagent molecule or it may be a dye or reagent bound to antibodies, nucleic acid probes, lectins or other large carrier molecules that have a specific affinity for a component of the sample. Colorant reagent molecules include dye precursors which react to provide observable color or fluorescence.
The term “stain precursor” is used herein to refer to a carrier molecule having specific affinity for a compound which is unlabeled, i.e., does not include a dye or colorant reagent. In order to visualize the tissue, the stain precursor is complexed with a second labeled compound to form a multi-molecular conjugate. Such a stain conjugate may include a primary compound, e.g., a primary antibody having an affinity for a component of the tissue, which itself is not observed by conventional imaging techniques, and a secondary compound, e.g., a secondary antibody having affinity for the primary antibody, e.g., an anti-immunoglobulin antibody, which bears a dye or colorant reagent used to image the tissue or an enzyme which converts a colorless substrate into a colored substrate.
The term “component of the sample or tissue” is used herein to refer to the epitope or other r
Kerschmann Russell L.
Kuwahara Thomas
Odom Ronald
Reddington Mark
Clark & Elbing LLP
Resolution Sciences Corporation
Starsiak Jr. John S.
Warden Jill
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