TWI252912B - Histological processing of tissue and other material - Google Patents

Abstract

The invention features a method for enbloc 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

1252912 V. INSTRUCTIONS ^- BACKGROUND OF THE INVENTION

The method of the present invention relates to a square a microscopic examination for processing a tissue sample for visualization, and the production of the sample can be used as a sample. After the gradual replacement of the fine and the second, it is solidified and carried out in block organic matter with the substance - and the side is preserved and continuously osmized to shift toluene. Then it is. This extra. The microwave penetrates into the sample-package method of the visible tissue-like series, and the product 5 avoids the permeation step. In addition to the sample, in the conventional melting-hardened paraffin furnace, the light and electron microscopy have been used to penetrate the product. And the preparation of other materials, normally borrowed, chemically treated, resulting in a solid block (bl〇ck) buried therein. The tissue is first treated with fumarin, self-dissolving (self-decomposing) permeability, thereby enhancing the subsequent design of the paraffin by designing an optical microscopy by increasing all the moisture, and then, infiltrating The tissue is then placed around to produce a tissue-based glutaraldehyde used in the histological laboratory, or a solvent used to immobilize the solution to make the solution osmotic as a solvent, for example, in a submerged sample. Cooling to the chamber in a mold block (in the tissue 'to add to the fan;

In addition to paraffin, the plastic can also be methacrylate, but in any case, the tissue is treated as approximately. The moisture in the tissue is replaced by a bulk polymer. The polymer is then cured (polymerized), including in the general use of the resulting building blocks. The plastic oxy-resin polymer or related material is replaced by an organic solvent in the same manner as in paraffin, followed by liquefied other components, exposed to ultraviolet light, heat, or added by various means.

Page 4 1252912 V. INSTRUCTIONS (4) To deal with tissue, this substance binds to tissue at the molecular level. The term ''polar π' and ''hydrophilic'' as used herein is synonymously referring to a chemical substance that is highly water-soluble, while the noun π is non-polar and ', and anaerobic' refers to a chemical substance that is poorly water-soluble, but It is easily soluble in organic solvents and soluble in fats or lipids. The term "opaque agent" as used herein refers to a substance that is added to the embedded and infiltrated matrix to produce a very high absorbance which inhibits the image of the tissue, resulting in a depth exceeding a few microns in the matrix. Opaque agent

An example is Fat Brown RR (Solvent Brown 1 ; sigma Chemicals, St. Louis, MO) 〇 use of non-polar osmotic and embedding materials (eg epoxy or paraffin, where polar dyes show little solubility), with The tissue is stained with a polar dye to distribute the polar dye into the tissue to stain the hydrophilic component (eg, protein) and prevent the dye from entering the surrounding matrix to enhance the height of the contrast image. Alternatively, non-polar, fat-soluble (i.e., water-repellent) dyes can be used to stain the fat-rich structures (e.g., cell membranes) in the tissue. In such a situation, polar embedding materials such as ethylene glycol methacryl are used.

Acid S (Polysciences, Inc. Warrington, PA) to avoid diffusion of non-polar dyes from the tissue to the embedded matrix, thereby enhancing contrast in tissue images. One of the main reasons for the focus of the present invention is that the infiltrated and embedded matrix is not removed after the overall staining' and before some types of microscopy (including basal surface microscopy) imaging. Therefore, the use of these 1252912 V, invention description (5) technology is important for the production of high-contrast images, the image signal will be eliminated, that is, the source is derived from the embedding material. High contrast is essential. Because the choice is based on the use of the embedding matrix between the 叩 and the embedded matrix (for example, the dye of ethylene glycol 7*, with polarity (solid polyethylene glycol; Union Carbide Ac Acrylic 5 and Carbowax CT) ), therefore, the first integral dyeing place Danbury enters the surrounding matrix. Embedding matrix without dyeing agent = improved comparison between wood and 彳 avoidable tissue and surrounding matrix. ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, The substrate in a given matrix is empirically determined. For many times = ' can be used for any given data (for example, Merck Index) to determine the resolution of the solution to the valley can be used to reference U / v; 1.0 g / 100 ml, 0%), or liter: show grams or weight Molecular concentration. For example, the solubility of ¥高\表氏不/^克克浓度7-methylamino-2-methylphenazine hydrochloride, 4, 10 liters; 18% in anhydrous alcohol; 3.0% solubility in ethylene glycol; and 2 solubility in xylene (Merck Index, supra). Another dye, Eosin 1 ^ The following solubility: 44.0% in water; 2% in alcohol; 25.0% in ethyl ether (Cellosolve); 27.5% in ethylene glycol; Less than 0.1% in a toluene (Electr〇n Micr〇sc〇py SeieneesFQft one

1252912

V. Description of invention (6)

Washington, PA, 1 99 9 Table of Contents). The most important element of a method of the invention is that the solubility in the embedding solution is less than the solubility of the dye in the dye solution. The solubility of the dye in the phase 1 solution, the lower the percentage of solubility in the embedding solution, the higher the contrast. Preferably, this percentage is less than 5%, / more preferably less than 20% ' and optimally less than 1% or even 2%. °

Table 1 contains a series of dyes compatible with polar dye solutions, as well as related non-polar embedded matrices suitable for use in the present invention. Table 1 also contains a series of polar embedding matrices compatible with lipid-coated N i 1 e Red. These lists are for illustrative purposes and are not intended to limit the invention. It should be understood that 'other dyes, such as listed in C ο η η, H. J ·,, biological staining n (9th edition, The Williams and Wilkens Co., B a 11 imore 1 9 7 7 ) The dye may also be compatible with one or more of the embedded substrates listed below. Similarly, other embedding matrices may be compatible with, for example, the dyes provided in Table 1 or Conn (above). Table 1

Incompatible with polar dyeing solution, non-polar embedded matrix dyeing agent compatible with non-polar dyeing solution polar embedded matrix spheroidized spheroidal snail · brominated ethyl bond Araldite acridine right Epon blush Y Spurr Texas Red Fire Cotton (Cellodin) DAPI Newport Green Green TMFL Nile Red Carbowax Ethylene Glycol F-Based Acrylic Jun Yang Lai Animal Gelatin OTC

Example

Page 9 1252912 V. INSTRUCTIONS (7) Example 1 : A tissue preparation in which the protein in the sample is to be dyed as a whole, comprising the following ingredients, wherein the dye is highly water soluble and the embedded matrix is boring Aqueous: Dye: 2% aqueous solution of Eosin Y fluorescent dye (Si gma Chemicals, St. Louis, MO). Embedding/infiltrating matrix: a paraffin-based embedding matrix (Surgipath Medical Industries Inc., Richmond IL). Opaque Agent · Saturated Fat Brown RR (Solvent Brown 1 ; Sigma Chemicals, St. Louis, MO) 〇

An opaque substrate is prepared by dissolving an excess of opaque agent in a molten paraffin with moderate agitation. The mixture was allowed to stand for 1 hour in a continuously heated environment and then poured out. Tissue samples were stained by immersion in Eosin Y solution, subsequently treated with graded alcohol and dioxins, then infiltrated and embedded in an opaque matrix. The tissue blocks were visualized on a block surface microscope. Example 2:

A tissue preparation in which a nucleic acid in a sample is to be dyed, wherein the dye is highly water-soluble, and the embedding substrate is water-repellent, comprising the following components: Dye: 1% aqueous solution of propidium iodide dye ( Sigma ChemieaU, St. Louis, M0). Embedding/Infiltration Matrix: Epon Epoxy Resin Matrix (Polysciences, Inc. Warrington, PA) °

Epon is prepared according to the manufacturing manual. Tissue samples were stained by immersion in a propidium iodide solution, followed by fractionated alcohol and xylene

Page 10 1252912

Then, it is infiltrated and embedded in an epoxy resin matrix. The tissue blocks were visualized on a development microscope. Embodiment 3 A tissue preparation method in which a neutral lipid in a sample is dyed, comprising the following components: wherein the dye is low in water solubility, and the embedded matrix is highly water-soluble: dyeing agent: 1 % Nile red dye solution (M〇lecular Pr〇bes, Eugene, OR) 'dissolved in 2-methoxyethanol. Embedding/infiltrating matrix: complete concentration of ethylene glycol methacrylate catalyzed infiltration resin

(Polysciences, lnc· Warrington, PA). The tissue samples were placed in a dye solution in individual 1 〇 ml vials and spun for 24 hours. The infiltrated and stained samples were then transferred to 2 ml of embedded capsules with excess formulation, vortexed for 24 hours, centrifuged at 5,00 rpm (r pm ) for 10 minutes to allow the sample to precipitate easily and allow Polymerization at room temperature (about 1 hour). The blocks were visualized on a surface development microscope. references

Laboratory methods for histological techniques: p r e p a r e d b y t h e A r m e d Forces Institute of Pathology· ed· Prophet, Ε·Β·, Arrington J.B., Sob i n L.H. American Registry of Pathology, Washington, D.C., 1992.

Carson, F.L. Histology: A self-instructional

Text. ASCP Press, American Society of Pathologists, Chicago, 1990 o S h e e h a n, D · C ·, H r a p c h a k, B · B · Theory of histological techniques

1252912 V. INSTRUCTIONS (9) AND PRACTICE, 2nd ed., Battelle Press, Columbus 0H, 1980. U.S. Patent No. 4,9, 60, 3, 3, ''Development Recording Apparatus'. All patents and publications mentioned herein are incorporated herein by reference.

1084-3013-PFl.ptc Page 1 1252912 V. INSTRUCTIONS (2) = Learning catalyst. The plastic operation method has been cut by the optical and electronic microscopy: in each of the following: i: ί, the base block containing the tissue is placed in the microscopic examination of the slicer, and the slice is smashed on the glass slide. Place. Once fixed, rhyme dyeing, these dyes are marked with special, and the slices are then subjected to various dyes, etc., to the other, (,, _ 疋, 、, TP fields (eg, nucleic acid, lipid method. 'Processing these sections for immunohistochemistry. In addition, these methods are used to infiltrate and embed, and the body (4) (4) stains 'where the 'cut surface of the block itself is called an X: transmissive microscopy' or Base image. In the latter case, a sample that is developed by means of block microscopic examination, followed by 1 and 2) cold has been dyed with a fluorescent dye as a whole - a plastic polymer, 4^) Embedding 'this matrix is ubiquitous in image suppression, heavy opacity or other treatments' degree. This results in a thin, deeper than a few micrometers in the matrix, which is very similar to the conventional glass. The special filament method of the present invention comprises the following steps for the overall dyeing and embedding of a sample in a solution which is reversibly immersed in a dye solution containing a dye. To a component of the sample; and (b) to sample

Case No. 89102812

1252912 V. INSTRUCTION DESCRIPTION (3) Embedding into an embedded matrix wherein the solubility of the dye in the embedded matrix does not exceed 5% of the dye in the dyeing solution. Preferably, the embedding substrate is also a permeable material and may comprise an opaque substance. Preferably, the solubility of the dye in the embedding matrix is not more than 20% of that dissolved in the dyeing solution; more preferably, the dyeing agent is in the embedding matrix, and the degree of disproportion does not exceed that in the dyeing solution. 10% or even 2%. In other more specific embodiments, the sample is a biological tissue sample, such as a sample obtained from a human cadaver, or a sample obtained from a biopsy patient. _ The term "sample" as used herein refers to other substances that are to be colored. Prior to dyeing, the core:::+embedded tissue or = thickness is greater than 200 microns, and may be greater than the gong, Chu, or 篆. The term "fixed" as used herein refers to a chemical product. It can be stored, hardened and penetrated. ☆Liquid treatment of tissue samples The term "infiltration" as used herein refers to the treatment of tissue in one body, which can penetrate the liquid to the molecular sound f to make the sample hard.

As used herein, the noun "sentence S" mold, and a substance J two two fingers to infiltrate the tissue in a ~ 玉玉衣 (usually in the group, 'mox, sign, ^ to form a package block The embedding material therefore, in the 1 day material, then the hard character, and makes the cutting step easy to make the knife to provide hard support. The noun used here is "sliced" and can be fixed in the glass. Sgo other support ^ : 4 # + cutting, but the noun used here, dyed ', refers to a right ", color or fluorescent substance

Claims (1)

Case No. 89102812 1252912 6. Method of applying for a patent range sample, the party includes the following 1. An overall dyeing and embedding step: (a) immersing the sample in a dyeing solution containing a dye, the dye reversibly bonded to the a component of the sample; and (b) embedding the sample in an embedded matrix, wherein the dye does not dissolve in the embedded matrix by more than 50% of the dye solution. 2. The method of claim 1, wherein the embedding matrix is also a permeable material. 3. The method of claim 1, wherein the embedding substrate comprises an opaque substance. 4. The method of claim 1, wherein the dye is dissolved in the embedding matrix no more than 20% of the dye in the dyeing solution. 5. The method of claim 1, wherein the dye does not dissolve in the embedding matrix by more than 1% of the dye in the dyeing solution. 6. The method of claim 1, wherein the dye is dissolved in the embedding matrix by no more than 2% of the dye in the dyeing solution. 7. The method of claim 1, wherein the sample comprises biological tissue. 1084-3013-PFl.ptc Page 13