JP2002537554A - Histological processing of tissues and other substances - Google Patents

Abstract

  (57) [Summary] The present invention provides (a) a step of immersing a sample in a staining solution containing a dye that reversibly binds to a sample component; Characterized by a method of en bloc staining and embedding of the sample, comprising the step of embedding the sample in an embedding medium, wherein the sample is dissolved in an embedding medium.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION The present invention relates to a method of processing a tissue sample for imaging.

In current technology, for organ microscopy and other materials for both transmission microscopy, ie, visible light microscopy and electron microscopy, the preparation of solid blocks in which the sample is embedded This is usually done by subjecting the sample to a series of chemical treatments leading to the preparation of

In this method, the tissue first protects the formalin, glutaraldehyde, or sample from self-melting (self-disintegration), increasing the permeability of the sample and strengthening the sample, thereby enhancing the penetration of the next solution Chemically fixed by other substances that help to The stage of infiltration following immobilization has been devised to remove all moisture from the sample by increasing the concentration of organic solvents such as alcohols and xylenes and gradually proceeding the displacement. In the case of conventional light microscopy, the last step of the penetration process is to penetrate the tissue with dissolved paraffin. The temperature of the sample is then reduced to room temperature, whereby the infiltrated tissue solidifies. The hardened and infiltrated tissue is then placed in a mold and further paraffinized to create a tissue block.

[0004] In addition to paraffin, plastic is also widely used for making tissue blocks. This may be a methacrylate, epoxy polymer, or related material, but in any case will subject the tissue to a treatment generally similar to that used for the paraffin treatment. The water in the tissue is replaced by an organic solvent, which is then replaced by a liquid polymer. The mixture of polymer precursor components is coagulated (polymerized) by various methods, including exposure to ultraviolet light and heat, and by the addition of chemical catalysts. Plastic processing is compatible with processing for both light and electron microscopy.

[0005] In either of these methods, the block containing the tissue is sliced with a microtome. For light microscopy, sections are collected and placed on glass slides. Once protected on slides, sections are stained with a number of dyes that label specific parts of the cells (eg, nucleic acids, lipids, etc.) or processed for immunohistochemistry.

Alternatively, the entire sample is stained by immersion prior to penetration and embedding,
These methods have been adopted for block staining. Then, a section is cut from the block for transmission microscopy, or a cut surface of the block itself is imaged by an operation called block face microscopy. In the latter case, a surface imaging microscope (U.S. Pat. No. 4,960,330, "Image Recording Appa"
specimens that have been unblock stained with fluorescent dyes, including those that have been put to practical use in ratus), are subsequently heavily opaque, or otherwise tissue images arising from depths of several microns or more within the block And is embedded with a drug, typically a plastic polymer, that has been treated to allow the control of the drug. This results in a thin “temporary section” that is very similar to a conventional glass slide on which tissue sections are mounted.

SUMMARY OF THE INVENTION The present invention provides (a) a step of immersing a sample in a staining solution containing a dye that reversibly binds to a sample component; A method for unblock staining and embedding a sample, comprising embedding the sample in an embedding agent, wherein the dye dissolves in the embedding agent.

[0008] Preferably, the embedding medium is also a permeable substance, and may include an opaque substance. Preferably, no more than 20% of the dye when dissolved in the staining solution dissolves in the embedding medium. More preferably, 10% or less of the dye or 2% or less of the dye when dissolved in the staining solution dissolves in the embedding medium. In other preferred embodiments, the sample is a sample obtained from a human autopsy or a biological tissue sample from a patient undergoing a biopsy.

[0009] The term "sample" is used herein to refer to tissue or other material that is stained and embedded. Samples were not prepared by microtome prior to staining. The thickness of the sample is greater than 200 μm, and may be greater than 1 mm, 1 cm, or more.

[0010] The term "fixation" is used herein to refer to the treatment of a tissue specimen with a chemical solution that protects, cures, and permeates the sample.

The term “penetration” as used herein refers to treating tissue with a liquid or series of liquids that penetrate into the tissue to the molecular level and then change to a solid to harden the sample. Used to

[0012] The terms "embedding method" and "embedding" are used herein to refer to a substance that includes infiltrated tissue in a mold and is cured to form a block contained in the container ( (Usually the same as the osmotic agent) is used to refer to containing the tissue. The embedding material thus serves to provide a rigid support and to facilitate the cutting process.

The term “slice” is used herein to refer to cutting a thin section from a block that can then be mounted on a glass slide or other support.

The term “staining” is used herein to refer to treating a tissue with a colorant or fluorescent material that binds the tissue at the molecular level.

The terms “polar” and “hydrophilic” are used interchangeably herein to refer to a chemical that is highly water-soluble. On the other hand, the terms "non-polar" and "hydrophobic" are used to refer to chemicals that are poorly soluble in water but instead are readily soluble in organic solvents and fats or lipids.

The term “opaquer” as used herein refers to a substance added to embedding and penetrants that produces a very high absorbance that suppresses tissue images that result from depths of several microns or more within the block. Used to mean. Typical opacifier is Fat
Brown RR (Solvent Brown, Sigma Chemicals; St. Louis, MO).

The use of non-polar osmotic and embedding materials, such as epoxy resins and parafines, in which polar dyes exhibit little solubility, along with tissue staining with polar dyes, involves distributing the polar dyes to tissues and protein And dyes, and inhibits the penetration of dyes into surrounding drugs, which helps to enhance the construction of the image.

Alternatively, the non-polar, fat-soluble (ie, hydrophobic) dye is a lipid-rich structure in tissue,
For example, it can be used to stain cell membranes. In such situations, polar embedding substances such as glycol methacrylates (PolySciences, Inc, Warrington, PA) are used to prevent the diffusion of non-polar dyes from the tissue into the embedding medium, thereby providing a histological image. Enhances contrast.

One of the main reasons for the importance of the present invention is that in some types of microscopy, including block-face microscopy, the permeation and embedding agent, after unblock staining, prior to imaging Is not removed. Therefore, it is important for these techniques to produce high-contrast images that image signals from the embedding material itself should be removed. That is, high contrast between the sample and the embedding medium is essential. Therefore, glycol methacrylate and carbowax (solid polyethylene glycol, Union Ca
rbide Corp, Danbury, Conn.) to select a relatively hydrophobic dye that is suitable for use with polar embedding media, such as dyes from surrounding unblocked tissue samples. It is prevented from entering the implant. Keeping the embedding agent free of dye leads to improved contrast between the stained tissue and the surrounding embedding agent.

[0020] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

The solubility of any dye in detailed description is given agent can also be determined experimentally in any given drug. For many dyes, solubility can be determined using references such as, for example, the Merck Index. Solubility is expressed in percent (weight / volume; 1) by grams of solute / volume of solvent (g / 100 mL or g / L).
(00 g / 100 ml = 100%) or by molarity or by molarity. For example, neutral red (3-amino-7-dimethylamino-2-methylphenazine hydrochloride), which stains the Golgi apparatus, is 4.0% (0.4%) in water.
g / L), 1.8% in absolute ethanol, ethylene glycol
3.0%, showing virtually zero solubility in xylene (Merck index, supra). Another dye, eosin Y, shows the following solubility: 44.0% for water; 2.0% for ethanol, 25.0% for cellosolve; 27.5% for ethylene glycol
A solubility of less than 0.1% in xylene (Electron Microscopy Sciences, Fort Washington, PA, catalog 1999).

The most important component in the method of the present invention is that the solubility of the dye in the embedding liquid is lower than that of the dye in the staining liquid. The lower the percentage of solubility of the dye in the embedding fluid compared to the solubility in the staining fluid, the higher the contrast. Preferably, this percentage is less than 50%, more preferably less than 20%, most preferably less than 10%, or even less than 2%.

Table 1 contains a list of dyes that can be exchanged for polar staining solutions, and corresponding non-polar embedding agents suitable for the invention described herein. Table 1 also includes a list of polar embedding agents that can be exchanged for Nile Red, which stains lipids. These listings are for illustrative purposes and do not impose any restrictions. Conn H. J. "Biological Stain", 9th edition, The Williams and Wilke
Other dyes, such as those listed in ns Co., Baltimore 1977, are interchangeable with one or more embedding agents listed below. Similarly, other embedding agents may be interchangeable with the dyes described, for example, in Table 1 or Conn (supra).

[0024]

[Table 1]

EXAMPLES Example 1 A tissue preparation in which the protein in a sample must be unblock stained, the dye is very water-soluble and the embedding medium is hydrophobic. Contains elements.

Dye is 2% of Eosin Y fluorescent dye (Sigma Chemical, St. Louis, Mo.)
It is an aqueous solution. The embedding / penetration agent is a paraffin-based embedding agent (Surgipath Me
dical Industries Inc, Richmond, Illinois). Opacifier is saturated
Fat Brown RR (Solvent Brown 1; Sigma Chemicals, St. Louis, Mo.).

The opacifying agent is prepared by dissolving an excess of the opacifying agent in molten paraffin with moderate stirring. The mixture is allowed to settle for 1 hour with continuous heating,
Then it is decanted. Tissue samples are stained by immersion in eosin Y solution, then processed through graded concentrations of alcohol and xylene, and penetrated with an opacifier and embedded in the opacifier . The tissue block is imaged with a block face microscope.

Example 2: Tissue preparation in which the nucleic acid in the sample must be stained, where the dye is very water-soluble and the embedding medium is hydrophobic, comprises the following components:

The dye is a 1% aqueous solution of propidium iodide (Sigma Chemicals, St. Louis, Mo.). The embedding / penetration agent is Epon epoxy medium (Epon epoxy medium)
Polysciences, Inc., Warrington, PA).

Epon is prepared according to the manufacturer's instructions. Tissue samples are stained by immersion in a propidium iodide solution, followed by treatment with graded concentrations of alcohol and xylene, and penetrated with an epoxy agent and embedded in an epoxy agent. The tissue block is imaged with a surface imaging microscope.

Example 3 In a tissue preparation in which neutral lipids in a sample must be stained, a dye having low water solubility and an embedding medium having very high water solubility contains the following components: Including.

The dye was Nile Red stain (Molecular Probes, Eugene, Oreg.)
-1% solution in methoxyethanol. The embedding / penetration agent is full strength glycol methacrylate ester catalyzed permeation resin (JB-4, Polyscience
s, Inc, Warrington, PA).

The tissue samples are placed in the staining solution in each 10 mL vial and tumbled for 24 hours. The infiltrated and stained sample is then transferred to a 2 mL embedding capsule with excess drug, tumbled for 24 hours, centrifuged at 5000 rpm for 10 minutes to facilitate sample precipitation, and polymerized at room temperature. (About 1 hour) The blocks are imaged with a surface imaging microscope.

Reference : "Laboratory Methods in Histotechnology"
: Prepared by Military Pathology Research Institute. Prophet EB, Mills B., Arrington JB, Sobin LH, Ed., American Pathology Record, Washington DC, 1992.

Carson FL, “Tissue Engineering” (Histotechnology): A Self-Instructional Text, American Society of Clinical Pathology, American Pathologists Association, Chicago, 1990

Sheehan DC, Hrapchak BB, “Theory and Practice of Histotechnology”, Second Edition, Battell
e-publishing, Columbus, Ohio, 1980

US Pat. No. 4,960,330, “Image Recording Apparatus”

[0038] All patents and publications referred to herein are hereby incorporated by reference.

[Procedure amendment]

[Submission date] February 14, 2002 (2002.2.14)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Name of invention

[Correction method] Change

[Contents of correction]

Title: Histological treatment of tissue and other substances

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Claims (7)

[Claims] 1. A method for en bloc staining and embedding a sample, the method comprising: a) dissolving the sample in a staining solution containing a dye that reversibly binds to a component of the sample. Dipping, and b) embedding the sample in an embedding medium, wherein 50% or less of the dye is dissolved in the embedding medium as compared with the case of dissolving in the staining solution. 2. The method of claim 1, wherein the embedding is also an osmotic agent. 3. The method of claim 1, wherein the embedding agent comprises an opacifying agent. 4. The method of claim 1, wherein no more than 20% of the dye when dissolved in the staining solution is dissolved in the embedding medium. 5. The method of claim 1, wherein no more than 10% of the dye when dissolved in the staining solution is dissolved in the embedding medium. 6. The method of claim 1, wherein no more than 2% of the dye when dissolved in the staining solution is dissolved in the embedding medium. 7. The method of claim 1, wherein the sample comprises a biological tissue.