KR20200036849A - Paraffin shield coating for microscope slides - Google Patents

Abstract

The present invention relates to a shielding coating on a microscope slide. The present invention particularly relates to the selective application of a paraffin layer that shields biomaterials and inorganic chemical deposits from microbial attack and oxidation. More specifically, the present invention relates to the application of a paraffin layer on a microscope slide as an exposure mask on a precipitated biomaterial reactive target. The paraffin masking layer blocks biomaterials and chemical targets from exposure that can lead to degradation due to oxidation, and provides resistance to fungal growth while removing paraffin from the masking and coexisting tissue sections using conventional dye treatment steps.

Description

Paraffin shield coating for microscope slides

The present invention relates to a shielding coating on a microscope slide. The present invention particularly relates to the application of a paraffin layer to protect biomaterials and inorganic chemical deposits from microbial attack and oxidation. More specifically, the present invention relates to the application of a paraffin layer on a microscope slide as masking exposure to a precipitated biomaterial reactive target. The paraffin shielding layer blocks biomaterials and chemical targets from exposure to decomposition due to oxidation, provides resistance to fungal growth, while removing paraffin from shielding and coexisting tissue sections using conventional stain treatment steps do.

Paraffin wax is generally a white or colorless soft solid derived from petroleum, coal or oil shale, which consists of a mixture of hydrocarbon molecules having 20 to 40 carbon atoms. Paraffin wax is a solid at room temperature and begins to melt above 99 ° C (37 ° C). The boiling point is above 370 ° C (698 ° F). Typical applications of paraffin wax include lubrication, electrical insulation and candles; Dyed paraffin wax can be made with crayons. It is different from kerosene and other petroleum products, sometimes called paraffin.

In pathology laboratories, paraffin wax is used to impregnate tissue before cutting a thin sample of tissue. The water is removed from the tissue through the synergistic strength of the alcohol (75% to absolute) and the tissue is removed from either an organic solvent such as xylene or an aliphatic substitute such as xylol. Subsequently, the tissue is placed in paraffin wax for several hours, then cooled and solidified in a mold with the wax, and then the slices are cut into microtomes.

The insertion of tissue sections into paraffin is a common practice for long-term preservation of tissue sections. However, the application of paraffin as a thin coating layer in selected areas of the microscope slide has not been reported. The selected region can contain proteins, conjugated proteins, antibodies, peptide strands or proteins coated beads or other cellular material. Paraffin is known to contain antifungal and antibacterial agents, which essentially prevent oxidation of the antigenic site and degradation of acid / base in the air at the exposed site. Paraffin shielding coatings change the lifespan of biomaterials and chemical targets from 3 days to 5 days to 1 year to 2 years, providing useful product life for end users. Removal of the inserted paraffin is also a routine practice to expose tissue sections to subsequent immunohistochemistry (IHC) or hematoxylin and eosin (H & E) staining. If the same or similar paraffin formulation is used to mask other precipitated material on the same microscope slide, no further slide treatment should be performed before IHC or H & E staining begins.

Reference may be made to Chinese Patent Publication No. 204790174 (U), which presents a paraffin seal instead of a cover slip to seal the two inlets to the buried chamber in a glass slide. The closed chamber allows precise microscopic examination of the sample without the risk of damage to the sample by microscope or other treatment. This practice does not support direct encapsulation of biomaterials or IHC treatment, especially deparaffinization of tissue sections.

Generally, in one aspect of the present invention, a paraffin coating is applied on a biological material and an inorganic target.

In another aspect of the present invention, biomaterials and inorganic targets are deposited on a microscope slide, where a paraffin coating is selectively applied to pass over the precipitate and cover as a shield.

In another aspect of the invention, the coated microscope slide made is post-heated to melt and / or mix the paraffin particles with a monolithic surface coating that seals both the sediment and the slide surface around the sediment.

In another aspect of the invention, the paraffin coating can be applied by spray coating, screen printing, inkjet methods, pad printing and roller transfer printing, and the like.

In another aspect of the invention, coated paraffins increase the shelf life of tissue sections by preventing acid and / or base degradation at the antigenic sites and exposed sites in the air.

Other aspects of the invention are disclosed in the following description.

1 is a cross-sectional view of a paraffin masking layer selectively applied on biomaterials and chemical precipitates.

The invention may be more readily understood by reference to the following detailed description, which constitutes a part of the invention. It is to be understood that the invention is not limited to the specific apparatus, method, conditions or parameters described and / or illustrated herein, and that the terms used herein are for illustration only and are not intended to limit the claimed invention. In addition, the singular forms 'a', 'an' and 'the' used in the specification including the appended claims include pluralities, and reference to specific values should at least specify the specific values unless the content clearly indicates otherwise. Includes. Ranges can be expressed in other specific values, "about" or "approximately" when expressed in other embodiments. In addition, unless otherwise indicated, the dimensions and material properties mentioned herein are illustrative, not restrictive, and are intended to better understand the sample typicality of suitable utility, and modifications other than the stated values may also be applied to specific applications. Accordingly, it may be within the scope of the present invention.

The invention is not limited in its application to the details of construction and the arrangement of components described. In the following description, or as shown in the description below, or as shown in the drawings, other embodiments are possible and the invention can be practiced or carried out in various ways. Also, phrases and terms used for the purpose of description herein should not be regarded as limiting. Use of "comprising", "consisting of", "having", "containing", "related" and variations and additional items thereof.

Now, preferred embodiments of the present invention will be described in detail.

Tissue blocks and sections cut from the tissue block are injected with paraffin, which completely replaces the moisture content of the tissue's cellular structure. Paraffin essentially does not aid in bacterial and fungal growth, thus ensuring long-term stability of visceral biomaterials.

Target proteins precipitated on microscope slides, glass or plastic provide a rich food source for bacterial or fungal countermeasures. In addition, the antigenic sites of proteins are vulnerable to oxidation, substantially neutralizing the ability of the detection antibody to bind to the protein. Many of the subsequent reaction binding sites are hydroxyls, which can be damaged through reaction with acids and bases in the air. In general, slides containing protein deposits are stored below the temperature that supports microbial growth. However, these restrictions limit the effective use of sediment. In addition, the protein precipitation slide is packaged in a vacuum sealed container to prevent oxidative damage. Unprotected protein precipitation slides have an outdoor shelf life of between 2 and 5 days depending on ambient temperature and air pollution levels.

Targets that react intrinsically with the chemical structure of the inorganic colorant are also prone to oxidation and reaction with substances in the air. These color reactant targets include those for hematoxylin, eosin and most special colorant groups. The target of this colorant is designed to react with only one stain. Most biological targets do not provide this specificity, but chemical structures are possible. Accordingly, the present invention provides a solution by providing a method of selectively coating biomaterials and targets on any kind of microscope slide to protect them from any kind of degradation.

In one embodiment of the invention, paraffin is mixed with a solvent to change the state of the substance from solid to liquid at room temperature. Mixing uses paraplast X-tra, or equivalents such as xylene or green xylene substitutes: Aliphatic solvents, for example xylol, reduce viscosity and slow the solidification rate after precipitation. In other examples, the solvent may be selected from, but is not limited to, toluene, paint thinner, turpentine, or a 50:50 mixture of acetone and kerosene.

In another embodiment, the solid paraffin is melted at a temperature not exceeding the paraffin melting temperature of 75 ° C. until it becomes a liquid, and then an aliphatic solvent is slowly added until a saturation point is observed (solid forms). Cool the mixture to 45 ° C. and slowly add more aliphatic until it is completely clear.

In another embodiment, the paraffin coating described above coats a microscope slide coated with a special stain-reactive precipitate pre-applied to the biomaterial and adhesive, where the biomaterial is a protein, peptide, conjugated protein, protein coated bead, peptide coated bead, or Conjugated coated beads, and special colored reactive end groups that uniquely occupy a specific colored material that reacts with the applied antibody and secondary coloring reagent.

In other embodiments, the paraffin layer can be deposited on, but is not limited to, spray, inkjet precipitation, transfer printing (such as pad printing), screen printing and precipitation on an adhesive coated microscope slide. In all precipitation methods, the paraffin layer should be less than 5 μm thick so that all paraffins (shield and tissue section insertion) can be dissolved and removed during the paraffin removal step of the coloring process. Regardless of the precipitation process / method, the criteria for paraffin are as follows:

● preferably a thin layer of about 5 microns or less,

● Has a melting temperature of less than 60 ° C, preferably less than 56 ° C, dissolves when exposed to xylene or xylol (aliphatic replacement) solvents, and

● It has an ambient temperature hardness similar to that of inserted paraffin.

In another embodiment, the tissue block inserting the paraffin material is tissue prep and tissue prep 2 by Thermo Fisher, melting temperature 56 ° C, Leica's paraplast and paraplast plus, melting temperature 56 ° C, Leica's paraplast X-tra, the melting temperature may include 50 ℃ to 54 ℃, but is not limited thereto. Paraplast Extra (X-tra) specifically includes a phenolic antioxidant, butylated hydroxy toluene, to reduce oxidative degradation of proteins, peptides and inorganic targets.

In other examples, each is a mixture of purified paraffin, synthetic polymer and other materials to establish melting temperature, hardness and viscosity. What is embedded in paraffin does not support microbial growth.

In other embodiments, special coloring may include, but is not limited to: Alcian Blue, Alanine Blue-Orange G solution, Azan dye, Bielschowsky ) Silver Dye, Brown & Benn-Gramm Stain, Cresyl Violet, DAB, Fontana Masson, Gordon and Sweet's silver staining ), Grosset's methaneamine silver method, Hall's bilirubin dye, Jones methaneamine silver method, Luxol Fast Blue, Luxol Fast Blue-Cresyl Violet, Mucicarmine (Meyer's Method), Muller-Mowry Colloidal Iron, Orange G, Nuclear Fast Red, PAS with Diastase Digestion, Periodic Acid Schiff (PAS), Phosphorus Tungstic Acid, Hematoxylin, Picro Sirius Red, toluidine Toluidine Blue Acidified, Trichrome-Comoris One-Phase, Trichrome-Masson's, Victoria Blue, Von Kossa, Weissert's Resorcin from Wesert Fuchsin, Weigert's Iron Haematoxylin, Zell-Neelsen Method.

In other embodiments, the target is not limited as pigments such as black and white may be selected as colored precipitates and any pigment color may be included.

In other embodiments, microscope slides to which the above-mentioned paraffin coat can be applied may be selected from, but not limited to, glass, plastic or any polymeric material. In other embodiments, paraffin may be purified and free of water.

In another embodiment, the coated microscopic slides made are post-heated to melt and / or mix paraffin particles with a monolithic surface (integrated surface) coating that seals both the precipitate and the slide surface around the precipitate.

In another embodiment, the resulting microscope slide is heated after the paraffin has precipitated to push the solvent out of the paraffin to return to the cured state. It is recommended to use infrared light on the paraffin side of the slide. Melting paraffin from top to bottom allows the solvent to rise and evaporate from the paraffin without interruption. The results are shown in Figure 1 how molten paraffin ensures good sealing at the edge of the precipitate.

1 is a cross-sectional view of a paraffin masking layer selectively applied on biomaterials and chemical precipitates. The paraffin melted after being precipitated to drain the solvent needed to prepare the paraffin liquid and seal the edges with a slide and / or slide adhesive coating.

Example

The examples mentioned below are exemplified for the method of operation of the invention and should not be construed as limiting the scope of the invention.

[Example 1]

How to apply the spray:

Spray on surfaces with low air flow. Low liquid to air mixing is preferred. The mixture is sprayed onto the slide through the mask to cover the PRS target. Generally, 1 to 2 passes are required to form a layer less than 5 microns thick. The paraffin mixture reservoir and spray head are heated slightly above 56 ° C. so that the paraffin is fluid and remains fluid during the flight from the spray head to the slide. The spray coverage from the head is nominally 0.375 inches, but a mask can be used for smaller shielded areas. Post-infrared reheating ensures 100% sealing.

[Example 2]

Screen printing method:

The stainless steel screen will be heated by passing an electric current through the wires of the screen between two parallel sides. The temperature of the screen should be slightly lower than the paraffin melting temperature so that paraffin does not seep into the bottom surface of the screen. Basically, paraffin acts more as a paste than a liquid. PRS requires a reheat cycle to ensure 100% sealing.

[Example 3]

Inkjet method:

The inkjet head needs to have a heater integrated within the print head to keep the paraffin in a liquid state. The post-heating cycle of the slide ensures 100% sealing.

[Example 4]

Roller transfer printing method:

The heated roller pulls the paraffin film from the heated reservoir onto the roller. The rollers transfer much of the paraffin film onto the slide in the same way as painting the walls with a hairy roller. Post reheating of the slide ensures 100% sealing.

This application claims the priority of U.S. Provisional Application No. 62/520169 filed on June 15, 2017 and U.S. Provisional Application No. 62/520319 filed on June 15, 2017, the disclosure of each of which is incorporated by reference in its entirety. It is incorporated herein by reference.

Claims (14)

A microscopic slide having a paraffin shielding coating for biomaterials and / or inorganic precipitates on a microscope slide, wherein the paraffin shielding coating comprises:
(a) melting the solid paraffin at a temperature in the range of 60 ° C to 75 ° C until liquid paraffin melts;
(b) adding a solvent to the liquid paraffin obtained in step (a) until a saturated mixture is obtained;
(c) cooling the mixture obtained in step (b) at a temperature of 30 ° C to 33 ° C, then slowly adding a solvent until a clear paraffin liquid is obtained;
(d) applying the layer of the clear paraffin liquid obtained in step (c) on the biomaterial and / or inorganic precipitate on the microscope slide to form the paraffin shield coating on the entire or selective area of the microscope slide. To do; And
(e) evaporating the solvent from the paraffin shielding coating by applying infrared heat to restore it to a hardened and solid state; a microscope slide coated on the microscope slide. According to claim 1,
The microscopic slide characterized in that the solid paraffin is purified and free of moisture. The method of claim 1 or 2,
The solid paraffin microscope slide characterized in that it has a melting temperature of 50 ℃ to 60 ℃. The method according to any one of claims 1 to 3,
The paraffins are TissuePrep and TissuePrep 2 from Thermo Fisher having a melting temperature of 56 ° C, and Paraplast from Leica having a melting temperature of 56 ° C, and Microscope slide characterized in that it is selected from Paraplast plus, and Paraclast X-tra from Leica, which has a melting temperature of 50 ° C to 54 ° C. The method according to any one of claims 1 to 4,
The solvent is xylene, an aliphatic xylene substitute (e.g. xylol), toluene, paint diluent, turpentine, 50:50 mixture of acetone and kerosene, or the above Microscope slides, characterized in that they are selected from mixtures of the listed solvents. The method according to any one of claims 1 to 5,
The layer of step (d) is a microscope slide characterized in that it has a thickness of 1 to 5 microns, preferably 2 to 3 microns. The method according to any one of claims 1 to 6,
The biomaterial and / or inorganic precipitate is selected from tissue sections, protein-related samples, inorganic targets and imaging reference targets, or combinations thereof. The method of claim 7,
The protein-related sample is a microscope slide characterized in that it is selected from proteins, peptides, conjugated proteins, protein-coated beads, peptide-coated beads and conjugated-coated beads that react with the applied antibody and secondary staining samples. The method of claim 7,
The image reference target is a pigment colored precipitate, preferably a microscope slide characterized in that it is a black and white pigment. The method of claim 7,
The inorganic target is Alcian Blue, Alanine Blue-Orange G solution, Azan dye, Bielschowsky dye, Brown & Benn-Gram dye ( Gramm Stain, Cresyl Violet, DAB, Fontana Masson, Gordon and Sweet's silver staining, methamine silver method from Grosset, Billirubin from Hall Dyes, Jones Methaneamine Silver Method, Luxol Fast Blue, Luxol Fast Blue-Cresyl Violet, Mucicarmine (Meyer's Method), Muller-Mowry Colloidal Iron, Orange G , Nuclear Fast Red, PAS using Diastase Digestion, Periodic Acid Schiff (PAS), Phosphorus Tungstic Acid, Hematoxylin, Picro Sirius Red, Toluidine Blue Acidification (Toluidine Blue) Acidified), Trichrome-Comory One-Step, Tri Trimrome-Masson's, Victoria Blue, Von Kossa, Weigert's Resorcin Fuchsin, Weigert's Iron Haematoxylin, Gel )-Microscopic slide characterized by being a compound that reacts with a dye selected from the Neelsen Method. The method according to any one of claims 1 to 10,
In step (d), the clear paraffin liquid obtained in step (c) can be applied through a spray coating method, an inkjet precipitation method, a transfer printing method, a screen printing method, and a vapor deposition method. The method according to any one of claims 1 to 11,
The microscope slide is a microscope slide, characterized in that the glass or plastic slide. According to claim 1,
The paraffin shielding coating protects biomaterials and / or inorganic deposits on the microscope slides from oxidation and / or microbial attack, and protects inorganics from reactions that generate oxidation and / or acid and / or corrosive substances delivered to the air. Microscope slide characterized in that it is used to protect. According to claim 1,
The paraffin masking coating is used to increase the life of the biomaterial and / or inorganic precipitate on the microscope slide.

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