WO2000049383A1 - Combined en bloc staining and embedding process - Google Patents

Abstract

The invention features a method for en bloc staining and embedding a sample, including the steps of (a) immersing the sample in a staining solution containing a dye that binds reversibly to a component of the sample, and (b) embedding the sample in an embedding medium, wherein the dye is no more than 50 % as soluble in the embedding medium as it is in the staining solution.

Description

COMBINED EN BLOC STAINING AND EMBEDDING PROCESS BACKGROUND OF THE INVENTION

The invention relates to a method of processing a tissue sample for imaging. In present day practice, the preparation of organic tissue samples and other material for transmission microscopy, both visible light and electron microscopy, is normally carried out by subjecting the samples to a series of chemical treatments culminating in the production of a solid block in which the sample is embedded.

In this process the tissue is first chemically fixed with formalin, gluteraldehyde, or other materials which serve to preserve the sample from autolysis (self- degradation), and to render the sample rigid as well as to increase its permeability, thereby enhancing the infiltration of subsequent solutions. The infiltration steps that follow fixation are designed to remove all of the water from the sample through progressive replacement with increasing concentrations of organic solvents such as alcohol and xylene. In the case of conventional light microscopy, the last step in the infiltration process is to infiltrate the tissue with melted paraffin. The temperature of the sample is then lowered to room temperature, whereupon the infiltrated tissue solidifies. The hardened, infiltrated tissue is then positioned in a mold and surrounded by additional paraffin to produce a tissue block.

Apart from paraffin, plastic is also widely employed for producing tissue blocks. This may be methacrylate, epoxy polymer, or related material, but in any case the tissue is subjected to a process roughly similar to that employed in paraffin processing. The water in the tissue is replaced by organic solvents, which are in turn replaced by liquid polymer. The mixed precursor components of the polymer are made to solidify (polymerize) by various means, including exposure to ultraviolet light, heat, or by addition of a chemical catalyst. Plastic processing has been adopted for both light and electron microscopy. For each of these methods, 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, alternatively, are processed for immunohistochemistry.

Alternatively, methods have been introduced for en bloc staining, wherein the entire sample is stained by immersion, prior to being subjected to infiltration and embedment. 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. patent number 4,960,330, "Image Recording Apparatus"), a sample that has been stained en bloc with fluorescent dyes is subsequently infiltrated by and embedded in a medium, commonly a plastic polymer, 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.

SUMMARY ΩE THE INVENTION

The invention features a method for en bloc staining and embedding a sample, including the steps of (a) immersing the sample in a staining solution containing a dye that binds reversibly to a component of the sample, and (b) embedding the sample in an embedding medium, wherein the dye is no more than 50% as soluble in the embedding medium as it is in the staining solution.

Preferably, the embedding medium is also the infiltrating substance, and may include an opacification substance. Preferably, the dye is no more than 20% as soluble in the embedding medium than it is in the staining solution; and more preferably, the dye is no more than 10% or even 2% as soluble in the embedding medium than it is in the staining solution. In other preferred embodiments, the sample is a biological tissue sample, such as a sample obtained by human autopsy or from a patient from whom a biopsy has been obtained.

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 is greater than 200 microns, and can be greater than one millimeter, one centimeter, or more.

The term "fixation" is used herein to refer to the treatment of tissue specimens with a chemical solution that preserves, hardens, and permeabilizes the sample. The term "infiltration" is used herein to refer to treating the tissue with a liquid or series of liquids that penetrate throughout the tissue to the molecular level and are then transformed into a solid in order to render the sample rigid.

The terms "embedding" and "embedment" are used herein to refer to containing the infiltrated tissue in a mold and surrounding it with a substance (usually the same as the infiltrating substance) which is then hardened to form an encasing block. The embedding substance thus serves to provide rigid support and to facilitate the cutting process.

The term "sectioning" is used herein to refer to cutting from the block thin slices that may then be mounted on glass slides or other support. The term "staining" is used herein to refer to treating tissue with a colored or fluorescent substance that associates with the tissue on the molecular level.

The terms "polar" and "hydrophilic" are used synonymously herein to refer to chemical substances which are strongly water-soluble, while the terms "non-polar" and "hydrophobic" refer to chemical materials which are poorly water-soluble, but instead are readily soluble in organic solvents, and in fats or lipids.

The term "opacification agent" is used herein to mean a substance added to the embedding and infiltration medium to produce a very high absorbance which suppresses images of tissue originating from more than a few microns deep in the block. An exemplary opacification agent is Fat Brown RR (Solvent Brown 1 ; Sigma Chemicals, St. Louis, MO).

The use of non-polar infiltration and embedding materials such as epoxy or paraffin, in which polar dyes display little solubility, together with staining the tissue with polar dyes, allows the polar dyes to be partitioned into the tissue to stain hydrophilic components such as proteins and inhibits the dyes from entering the surrounding medium, promoting a high contrast image.

Alternatively, non-polar, fat-soluble (i.e., hydrophobic) dyes can be employed to stain lipid-rich structures in tissue, for example cell membranes. In such a situation, polar embedding materials such as glycol methacrylate (Polysciences, Inc. Warrington, PA) are employed to prevent diffusion of the non-polar dyes from the tissues into the embedding medium, thereby enhancing contrast in the tissue image.

A primary reason for the invention's importance is that the infiltration and embedding medium is not removed following en bloc staining and prior to imaging in some types of microscopy, including block face microscopy. Therefore it, is important for the creation of high contrast images using these techniques that image signals originating from the embedding material itself be eliminated; i.e., high contrast between the sample and the embedding medium is essential. Thus, because of the selection of relatively hydrophobic dyes for use with polar embedding media such as glycol methacrylate and Carbowax (solid polyethylene glycol; Union Carbide Corp., Danbury CT), dyes from the tissue sample, previously stained en bloc, are prevented from entering the surrounding medium. The maintenance of embedding medium free from dye leads to improved contrast between the stained tissue and the surrounding medium.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DETAILED DESCRIPTION The solubility of any dye in a given medium can be experimentally determined for any given medium. For many dyes, solubility can be determined using references such as, for example, the Merck Index. Solubility can be expressed in terms of grams of solute/volume of solvent (g/lOOmL or g/L), as a percent (w/v; lOOg/lOOmL = 100%), or in terms of molarity or molality. For example, neutral red (3-amino-7-dimethylamino-2-methylphenazine hydrochloride), which stains the golgi apparatus, exhibits 4.0% (0.4g/L) solubility in water, 1.8% in absolute ethanol, 3.0% in ethylene glycol, and practically no solubility in xylene (Merck index, supra). Another dye, Eosin Y, has the following solubility: 44.0% in water; 2.0% in ethanol; 25.0%) in Cellosolve; 27.5% in ethylene glycol; and less than 0.1 % in xylene (Electron Microscopy Sciences, Fort Washington, PA, 1999 catalog). The most important element of the method of the invention is that the dye solubility in the embedding solution is less than the dye solubility in the staining solution. The lower the percentage of solubility in the embedding solution compared to that in the staining solution, the higher the contrast. Preferably, this percentage is less than 50%, more preferably less than 20%, and most preferably less than 10%, or even 2%.

Table 1 contains a list of dyes compatible with polar staining solutions, and corresponding non-polar embedding media suitable for the invention described herein. Table 1 also contains a list of polar embedding media compatible with Nile Red staining of lipids. These lists are for illustrative purposes, and are not limiting in any way. It is understood that other dyes, such as those listed in Conn, H.J. "Biological Stain" 9th ed., The Williams and Wilkens Co., Baltimore 1977, may be compatible with one or more embedding medium listed below. Similarly, other embedding media may be compatible with the dyes, provided, for example, in Table 1 or in Conn (supra).

ABLE 1

EXAMPLES Example 1 :

A tissue preparation where proteins in the sample are to be en bloc stained contains the following components, in which the dye is highly water soluble and the embedding medium is hydrophobic:

Dye: 2% aqueous solution of eosin Y fluorescent stain (Sigma Chemicals, St. Louis, MO). Embedding/infiltration medium: paraffin-based embedding medium

(Surgipath Medical Industries Inc., Richmond IL). Opacification agent: saturated Fat Brown RR (Solvent Brown 1 ; Sigma Chemicals, St. Louis, MO)

Opacified medium is prepared by dissolving an excess of opacification agent in molten paraffin under moderate stirring. The mixture is allowed to settle for one hour under continued heating, and then decanted. Tissue samples are stained by immersion in the eosin Y solution, and subsequently processed through graded alcohols and xylene, then infiltrated with and embedded in the opacified medium. Tissue blocks are imaged on block face microscope. Example 2:

A tissue preparation where nucleic acids in the sample are to be stained, in which the dye is highly water soluble and the embedding medium is hydrophobic, contains the following components: Dye: 1% aqueous solution of propidium iodide stain (Sigma Chemicals, St.

Louis, MO). Embedding/infiltration medium: Epon epoxy medium (Polysciences, Inc., Warrington, PA).

Epon is prepared according to manufacturer's instructions. Tissue samples are stained by immersion in the propidium iodide solution, and subsequently processed through graded alcohols and xylene, then infiltrated with and embedded in the epoxy medium. Tissue blocks are imaged on a surface imaging microscope.

Example 3:

A tissue preparation method where neutral lipids in the sample are to be stained, contains the following components, in which the dye has low water solubility and the embedding medium is highly water soluble:

Dye: 1% solution of Nile Red stain (Molecular Probes, Eugene, OR), dissolved in 2-methoxyethanol. Embedding/infiltration medium: Full strength glycol methacrylate catalyzed infiltration resin (JB-4, Polysciences, Inc., Warrington, PA) Tissue samples are placed in the stain solution in individual lOmL vials and mixed by rotation for 24 hours. The infiltrated and stained samples are then transferred to 2mL embedding capsules with an excess of formulation, mixed by rotation for 24 hours, centrifuged at 5,000 m for 10 minutes to facilitate settling of the sample, and allowed to polymerize at room temperature (approximately 1 hour). The block is imaged on a surface imaging microscope.

REFERENCES

Laboratory Methods in Histotechnology: Prepared by the Armed Forces Institute of Pathology, ed. Prophet, E.B., Mills B., Arrington J.B., Sobin L.H. American Registry of Pathology, Washington, D.C. 1992

Carson, F. L. Histotechnology: A Self-Instructional Text. ASCP Press,

American Society of Pathologists, Chicago 1990

Sheehan, D.C, Hrapchak, B.B. Theory and Practice of Histotechnology. 2nd ed. Battelle Press, Columbus OH 1980

U.S. patent # 4,960,330 "Image Recording Apparatus"

All patents and publications referenced herein are hereby incoφorated by reference.

What is claimed is:

Claims

aims 1. A method for en bloc staining and embedding a sample, said method comprising: a) immersing said sample in a staining solution containing a dye that binds reversibly to a component of said sample; and b) embedding said sample in an embedding medium, wherein said dye is no more than fifty percent as soluble in said embedding medium as it is in said staining solution.

2. The method of claim 1, wherein said embedding is also the infiltrating substance.

3. The method of claim 1, wherein said embedding medium includes an opacification agent.

4. The method of claim 1, wherein said dye is no more than twenty percent as soluble in said embedding medium as it is in said staining solution.

5. The method of claim 1, wherein said dye is no more than ten percent as soluble in said embedding medium as it is in said staining solution.

6. The method of claim 1, wherein said dye is no more than two percent as soluble in said embedding medium as it is in said staining solution.

7. The method of claim 1, wherein the sample comprises biological tissue.