Sample compartmentalization module

Abstract

Described herein are tissue sample compartmentalization devices and methods for labeling and identifying one or more analytes in a tissue sample. In one embodiment, the device comprises a tissue sample compartmentalization module having a first member defining a first plurality of wells; and a second member defining a second plurality of wells, the second member being pivotably connected to the first member, wherein the second member is configured to engage with the first member to form a closed configuration, or to pivot relative to the first member between an open configuration and a closed configuration, wherein in the closed configuration, the first plurality of wells and the second plurality of wells are aligned and form a plurality of sample compartments. The tissue sample compartmentalization module provides individual sample environments for a sectioned tissue microscope slide.

Description

Background Technology

[0001] Microscopy of tissue samples stained with fluorescent dyes and / or immunostained (e.g., directly or indirectly stained with primary or secondary antibodies conjugated to fluorophores) provides valuable histological, cellular, and biomarker information. When multiple fluorescent reagents are used simultaneously on the same tissue sample, spectral permeation—the detection of fluorescence from neighboring fluorescent channels in the channel of interest—can hinder the identification of the actual target. One workaround is to limit the number of targets by staining the sample with a small number of spectrally separated fluorophores. However, multiplex detection is often necessary because tissue sample availability can be limited and tissue and detection reagents can be expensive. Furthermore, the process of preparing labeled tissue is laborious, especially when processing multiple samples in parallel. Even when using multiple spectrally overlapping fluorophores, additional control samples must be prepared with an appropriate single fluorescent reagent to computationally separate the target through linear unmixing.

[0002] What is needed is a device that can divide tissue into smaller compartments, thereby providing a separate sample environment for tissue samples cut on microscope slides or for other applications. Summary of the Invention

[0003] One embodiment described herein is a tissue sample compartmentation module comprising: a first member defining a first plurality of orifices; and a second member defining a second plurality of orifices; the second member being pivotally connected to the first member, wherein the second member is configured to engage with the first member to form a closed configuration, or to pivot relative to the first member between an open configuration and a closed configuration, wherein in the closed configuration, the first plurality of orifices and the second plurality of orifices are aligned. In one aspect, the tissue sample compartmentation module further includes a locking arm coupled to the second member, the locking arm being configured to rotate relative to the second member, wherein in the closed configuration, the locking arm is configured to engage a portion of the first member to secure the first and second members together. In another aspect, the locking arm defines a central channel, wherein in the closed configuration, the central channel of the locking arm is configured to receive a portion of the first member. In yet another aspect, the tissue sample compartmentation module further includes an insert defining a plurality of third orifices, wherein in the closed configuration, the insert is positioned between the first and second members, and the third plurality of orifices are aligned with the first plurality of orifices and the second plurality of orifices.

[0004] Another embodiment described herein is a method for labeling one or more analytes in a tissue sample, the method comprising: (a) placing a substrate containing the tissue sample onto a first member of a tissue sample compartmentation module described herein; (b) aligning the tissue sample with the first member; (c) placing an insert on the tissue sample such that a plurality of third wells defined by the insert are aligned with a plurality of first wells defined by the first member; (d) engaging or pivoting a second member relative to the first member to clamp the insert and the tissue sample between the first and second members and aligning the plurality of second wells with the plurality of third wells; (e) securing the first and second members together by locking arms to seal the insert to the tissue sample; and (f) incubating a solution containing affinity molecules in the aligned second and third wells, wherein the affinity molecules label analytes in the tissue sample. In one aspect, the sample is a tissue sample from a subject selected from: humans, non-human primates, rats, mice, guinea pigs, rabbits, pigs, cattle, sheep, goats, horses, dogs, cats, fish, birds, reptiles, amphibians, insects, plants, fungi, bacteria, or combinations thereof. In another aspect, the analyte includes one or more of metabolites, proteins, nucleic acids, carbohydrates, or lipids. In yet another aspect, the method further includes labeling two or more different analytes, wherein each aligned second and third well includes a solution containing a different affinity molecule for each analyte. In yet another aspect, the affinity molecule is an antibody, a portion of an antibody, an antibody-like molecule, a ligand receptor, a ligand of a receptor, a member of a coupling pair, an aptamer, or an antigen. In yet another aspect, the affinity molecule is conjugated to a fluorescent molecule, conjugated to an enzyme, bound to another affinity molecule conjugated to a fluorescent molecule, or bound to another affinity molecule conjugated to an enzyme. In yet another aspect, the substrate is a microscope slide.

[0005] Another embodiment described herein is a method for compartmentalizing a tissue sample for labeling two or more analytes in the tissue sample, the method comprising: (a) placing a substrate containing the tissue sample onto a first member of the tissue sample compartmentalization module described herein; (b) aligning the tissue sample with the first member; (c) placing an insert on the tissue sample such that a plurality of third wells defined by the insert are aligned with a plurality of first wells defined by the first member; (d) engaging or pivoting a second member relative to the first member to clamp the insert and the tissue sample between the first and second members and aligning the plurality of second wells with the plurality of third wells; and (e) securing the first and second members together by a locking arm thereby sealing the insert to the tissue sample. In one aspect, the sample is a tissue sample from a subject selected from: humans, non-human primates, rats, mice, guinea pigs, rabbits, pigs, cattle, sheep, goats, horses, dogs, cats, fish, birds, reptiles, amphibians, insects, plants, fungi, bacteria, or combinations thereof. In another aspect, the substrate is a microscope slide. In another aspect, step (c) further includes making the plurality of first holes defined by the first member overlap with the tissue sample as much as possible.

[0006] Another embodiment described herein is a method for analyzing a tissue sample for multiple analytes, the method comprising: (a) placing a substrate containing the tissue sample onto a first member of the tissue sample compartmentation module described herein; (b) aligning the tissue sample with the first member; (c) placing an insert on the tissue sample such that a plurality of third wells defined by the insert are aligned with a plurality of first wells defined by the first member; (d) engaging or pivoting a second member relative to the first member to clamp the insert and the tissue sample between the first and second members and aligning the plurality of second wells with the plurality of third wells; (e) securing the first and second members together by locking arms to seal the insert to the tissue sample; and (f) incubating a solution containing affinity molecules in the aligned second and third wells, wherein the affinity molecules label the analytes in the tissue sample; and (g) detecting a signal from each affinity molecule bound to the plurality of analytes, thereby detecting the presence or amount of each of the plurality of analytes. Alternatively, detection may be performed using a light or fluorescence microscope, a charge-coupled device (CCD) camera or imager, a phosphorescent imager, or a combination thereof. On the other hand, the sample is tissue selected from subjects including: humans, non-human primates, rats, mice, guinea pigs, rabbits, pigs, cattle, sheep, goats, horses, dogs, cats, fish, birds, reptiles, amphibians, insects, plants, fungi, bacteria, or combinations thereof. On the other hand, multiple analytes include one or more of metabolites, proteins, nucleic acids, carbohydrates, or lipids. On the other hand, the affinity molecule is an antibody, a portion of an antibody, an antibody-like molecule, a ligand receptor, a ligand of a receptor, a member of a coupling pair, an aptamer, or an antigen. On the other hand, the affinity molecule is conjugated to a fluorescent molecule, conjugated to an enzyme, bound to another affinity molecule conjugated to a fluorescent molecule, or bound to another affinity molecule conjugated to an enzyme.

[0007] On the other hand, the affinity molecule is conjugated with an enzyme. On the other hand, the enzyme is horseradish peroxidase. On the other hand, the detected signal comes from a tyrosamide conjugate. On the other hand, the tyrosamide conjugate comprises one or more of Alexa Fluor 350, 488, 546, 588, 594, 647 or 750 tyrosamide reagents or biotin-XX tyrosamide reagents.

[0008] Another embodiment described herein is a kit comprising: one or more tissue sample compartmentation modules as described herein; one or more affinity molecules; one or more signal generating reagents; optionally, one or more reagents, including deparaffinizing reagents, enzyme activating reagents, antigen retrieval reagents, sample dilution reagents, reagent dilution buffers, blocking reagents, or endogenous enzyme activity blocking reagents; and optionally, one or more containers, packaging, instructions for use, Material Safety Data Sheets (MSDS), reference or control tissue samples, and reference or control targets. Attached Figure Description

[0009] To describe how the above and other advantages and features can be obtained, a more specific description of the subject matter briefly described above will be presented by reference to specific embodiments shown in the accompanying drawings. It should be understood that these drawings depict only typical embodiments and are therefore not intended to limit the scope; the embodiments will be described and explained with additional specificity and detail using the drawings.

[0010] Figures 1A to 1B An example of spatial transcriptomics is shown. Figure 1A The spectral emission curves of nine fluorophores are shown, and the spectral overlap of different dyes (DAPI, eFluor 506, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 555, eFluor 615, Alexa Fluor 647, Alexa Fluor 700, Alexa Fluor 750) is shown. Figure 1B This image shows composite images of spectral mixing (left) and unmixing (right) of human tonsil tissue samples stained with nine fluorophores.

[0011] Figure 2 A flowchart illustrates the linear unmixing process for nine-fold samples. Multiple tissue samples were imaged using nine fluorescent dyes. Ten monochromatic control slides were prepared to extract the spectral curves of each fluorophore and tissue autofluorescence, from which the unmixing matrix was generated. The unmixing matrix was used to calculate the relative contribution of each fluorophore to each pixel of the mixed nine-fold image, producing the unmixed image.

[0012] Figure 3 An exemplary illustration shows a compartmentalized tissue sample on a microscope slide. The tissue section is fixed to the slide and divided into multiple sample regions. In the illustration, the compartmentation is a 4×4 array. The shape of the tissue sample controls the number of available compartments. In this example, only eight individual sample regions are compartmentalized within the tissue.

[0013] Figures 4A to 4BAn exemplary prototype organizational compartmentalization module is shown. Figure 4A The images show typical-sized tissue samples drawn with a blue marker pen, with two tissues drawn on each microscope slide. Figure 4B Showing attachment from Figure 4A Two prototype compartmentalization devices on a glass slide.

[0014] Figures 5A to 5E Fluorescence microscopy data obtained using a prototype tissue sample compartmentation device with three fluorescent dyes and an unstained control are presented. Figure 5A The compartmentalized tissue samples were shown, indicating three dyes (eF506, AF532, AF488) and an unstained control. Figures 5B to 5E Spectral data for eF506, AF532, AF488, and the unstained control are presented, as well as comparisons with standard methods (staining whole tissue sections without compartmentalization).

[0015] Figures 6A to 6C An example of an embodiment of the tissue sample compartmentation module is shown. Figure 6A This is a perspective view of the module whose configuration is displayed. Figure 6B This is a perspective view of the modules displayed in a closed configuration. Figure 6C This is a top view of the module in a closed configuration, showing multiple aligned holes.

[0016] Figures 7A to 7D The process workflow for using the tissue sample compartmentation module is demonstrated. Figure 7A A microscope slide is shown alongside tissue sections. Figure 7B Two tissue sample compartmentation modules in an open configuration are shown, located at the center of each tissue slice to maximize the available tissue. Figure 7C The module is shown in its closed and locked configuration and has been subjected to immunohistochemical (or other) assays. Figure 7D The removed module is shown, in which the sample is imaged using fluorescence microscopy and spectral extraction is performed as needed.

[0017] Figures 8A to 8B Fluorescence microscopy data for two fluorophores (AF488, AF555) obtained using a tissue sample compartmentation module, as compared with standard methods, are presented.

[0018] Figures 9A to 9D The process workflow for using the tissue sample compartmentation module with individual cell cultures is demonstrated. Figure 9A Microscope slides showcasing cell-attached surface coatings. Figure 9B The cell suspensions seeded in each compartment created by the tissue sample compartmentation module are shown. Figure 9CExperiments (e.g., drug treatment, immunofluorescence staining) were performed in various sample environments within the tissue sample compartmentation module. Figure 9D The microscope slides are shown ready for analysis after the tissue sample compartmentation module has been removed. Detailed Implementation

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclature used in conjunction with the microscopy, biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, and protein and nucleic acid chemistry and techniques described herein is well-known and commonly used in the art. In the event of any conflict, this disclosure (including the definitions) shall prevail. Exemplary methods and materials are set forth below, but similar or equivalent methods and materials may be used when implementing or testing the embodiments and aspects described herein.

[0020] As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “carrier,” “peptide,” and “protein” have common meanings that can be understood by a biochemist of ordinary skill in the art. Standard single-letter nucleotides (A, C, G, T, U) and standard single-letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.

[0021] As used herein, terms such as “include,” “including,” “contain,” “containing,” and “having” mean “comprising.” This disclosure also covers other embodiments, whether explicitly stated or not, that may “comprising”, “consistently comprise,” or “comprise” the embodiments or elements described herein. As used herein, “comprising” is an open-ended term and does not exclude the presence of other elements or method steps not expressly listed. As used herein, “consistently comprise” limits the scope of the claims to the specified materials or steps, and other content that does not substantially affect the basic and novel characteristics of the claimed invention. As used herein, “comprises” excludes any element, step, or ingredient not expressly recited in the claims.

[0022] As used herein, the terms “a,” “an,” “the,” and similar terms used in the context of this disclosure (especially in the context of the claims) should be interpreted to cover both the singular and the plural, unless otherwise stated herein or the context clearly contradicts this. Furthermore, unless otherwise stated, “a,” “an,” or “the” means “one or more.”

[0023] As used in this article, the term "or" can indicate both a parallel relationship (and) and a selective relationship (or).

[0024] As used in this article, the term "and / or" refers to both a parallel relationship (and) and a selective relationship (or).

[0025] As used in this article, the term “substantially” means to a great or significant degree, but not entirely.

[0026] As used herein, when the term “about” or “approximately” is applied to one or more values ​​of interest, it refers to a value similar to the stated reference value, or a value within an acceptable margin of error for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as limitations of the measurement system. In one aspect, the term “about” means any value, including integer and fractional parts, within a variation of up to ±10% of the value modified by the term “about.” Alternatively, according to convention in the art, “about” may also mean within a range of 3 or more standard deviations. Alternatively, such as for biological systems or processes, the term “about” may indicate a range of variation within a certain order of magnitude of a numerical value, in some embodiments within a range of 5 times, and in some embodiments within a range of 2 times. As used herein, the symbol “~” signifies “about” or “approximately.”

[0027] All ranges disclosed herein include the two endpoints as discrete values ​​and all integers and fractions specified within that range. For example, a range of 0.1 to 2.0 includes 0.1, 0.2, 0.3, 0.4, ..., 2.0. If the endpoints are modified by the term “about,” the specified range is extended by a variation of up to ±10% of any value (including the endpoints) within that range or within three or more standard deviations, or as defined above for “about.”

[0028] As used herein, the term “active ingredient” or “active pharmaceutical ingredient” refers to a pharmaceutical preparation, active ingredient, compound or substance, composition or mixture thereof that provides generally beneficial pharmacological effects.

[0029] As used herein, the terms “control” or “reference” are used interchangeably. A “reference” or “control” level can be a predetermined value or range used as a baseline or benchmark for evaluating measurement results. “Control” also refers to a control experiment or control cells.

[0030] As used in this article, “antibody” refers to an immunoglobulin or a fragment thereof, and encompasses any polypeptide containing an antigen-binding site, regardless of its source, manufacturing method, or other characteristics.

[0031] As used in this article, “analyte” or “antigen” refers to any substance that is identified by antibodies or other detection methods.

[0032] As used herein, “detectable label” refers to any molecule that can be detected directly or indirectly to reveal the presence of a target in a sample. Directly detectable labels can be used. A directly detectable label can be detected on its own without the need for additional molecules. Examples include fluorescent dyes, radioactive substances, and metal particles. Indirectly detectable labels can be used, which require one or more additional molecules. Examples include enzymes that affect the color change of a suitable substrate, and any molecule that can be specifically recognized by or react with another substance carrying the label. Other examples of indirectly detectable labels therefore include antibodies, antigens, nucleic acids and nucleic acid analogs, ligands, substrates, and haptens.

[0033] Examples of detectable markers that may be used herein include, but are not limited to, fluorophores, chromophores, electrochemiluminescent labels, bioluminescent labels, polymers, polymer particles, microspheres or other solid surfaces, gold or other metal particles or heavy atoms, spin labels, radioisotopes, enzyme substrates, haptens, antigens, quantum dots, aminohexyl groups, pyrene, nucleic acids or nucleic acid analogs or proteins such as receptors, peptide ligands or substrates, enzymes, and antibodies (including antibody fragments). Some detectable markers may include “color markers” in which a target is detected by the presence or change of color in a sample. Examples of “color markers” include, but are not limited to, chromophores, fluorophores, chemiluminescent compounds, electrochemiluminescent labels, bioluminescent labels, and enzymes that catalyze color changes in substrates. More than one color may be used, for example, by attaching a distinguishable color marker to a single detection unit or by using multiple detection units, each carrying a different and distinguishable color marker.

[0034] As used herein, a “fluorophore” is a molecule that emits detectable electromagnetic radiation when excited by electromagnetic radiation of one or more wavelengths. Many fluorophores are known in the art and have been developed by chemists for use as detectable molecular markers, and can be conjugated to affinity molecules described herein.

[0035] As used herein, the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or inhibition of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0036] As used herein, the terms “recognize,” “recognition,” or “recognizing” refer to an event in which a substance (such as an affinity molecule) interacts with a target directly or indirectly in any way such that the interaction with the target can be detected by the affinity molecule. In some non-limiting examples, the probe may react with or bind directly to the target, or react with or bind indirectly to the target by directly binding to another substance, which in turn reacts with or binds directly to the target.

[0037] As used herein, the term "subject" refers to an animal. Typically, a subject is a mammal. A subject also refers to primates (e.g., humans, male or female; infants, adolescents, or adults), non-human primates, rats, mice, guinea pigs, rabbits, pigs, cattle, sheep, goats, horses, dogs, cats, fish, birds, reptiles, amphibians, insects, plants, fungi, bacteria, or archaea. In one embodiment, the subject is a primate. In one embodiment, the subject is a human.

[0038] As used in this article and interchangeably with "analyte", "target" refers to any substance present in the sample that can be detected.

[0039] Device

[0040] refer to Figure 6A An example of an embodiment of a tissue sample compartmentation module 100 is shown. Module 100 includes a first member 104 and a second member 108. The second member 108 is pivotally connected to the first member 104 via a pivot assembly 112. The pivot assembly 112 includes a first pivot member 116 coupled to the second member 108. The first pivot member 116 is received by a first slot 120 defined by the first member 104. The pivot assembly 112 is configured to facilitate the second member 108 relative to the first member 104 in a first, open configuration (…). Figure 6A (as shown) and the second, closed configuration ( Figure 6B The pivoting motion between (shown). In other words, the second member 108 is configured to rotate relative to the axis defined by the first pivot member 116.

[0041] The first member 104 includes a planar surface 124. A plurality of first holes 128 are arranged on the planar surface 124. The first holes 128 extend through the planar surface 124 and completely through the first member 104. Thus, the first member 104 defines the plurality of first holes 128. In the illustrated embodiment, the plurality of first holes 128 are arranged in a grid pattern. More specifically, the plurality of first holes 128 are arranged in a 5×4 grid pattern. However, in other examples of the embodiments, the plurality of first holes 128 can be arranged in any suitable or desired pattern. Furthermore, the plurality of first holes 128 are shown as having a square shape. In other examples of the embodiments, the plurality of first holes 128 can have any suitable polygonal shape (e.g., circle, triangle, rectangle, pentagon, etc.).

[0042] The first member 104 includes a pair of first protrusions 132 extending away from the planar surface 124. Each of the first protrusions 132 defines a slot 120. The first member 104 also includes a second protrusion 136. The second protrusion 136 extends away from the planar surface 124 such that the planar surface 124 is recessed relative to the protrusions 132, 136. In other words, the first member 104 is offset relative to the second member 108. The protrusions 132, 136 are positioned at opposite ends of the first member 104. The protrusions 132, 136 also serve as guides to facilitate the alignment of the microscope slide on the planar surface 124 of the first member 104.

[0043] Now for reference Figure 6B The second member 108 includes a plurality of second holes 140. The second holes 140 extend completely through the second member 108. Therefore, the second member 108 defines a plurality of second holes 140. Each of the plurality of second holes 140 is perpendicularly aligned with a first hole of a plurality of first holes 128. Thus, the plurality of second holes 140 are arranged in a configuration similar to the first holes 128. In the illustrated embodiment, the plurality of second holes 140 are arranged in a grid pattern, and more specifically, in a 5×4 grid pattern. However, in other examples of the embodiments, the plurality of second holes 140 can be arranged in any suitable or desired pattern such that each second hole 140 is perpendicularly aligned with its associated first hole 128. Furthermore, the plurality of second holes 140 are shown as having a square shape. In other examples of the embodiments, the plurality of second holes 140 can have any suitable polygonal shape (e.g., circle, triangle, rectangle, pentagon, etc.). The shape of the second holes 140 is preferably the same as the shape of the first holes 128.

[0044] The second member 108 includes a locking arm 144. The locking arm 144 is positioned at the end of the second member 108 opposite to the pivot member 116. The locking arm 144 includes a second pivot member 148. The second pivot member 148 is received by a second slot 152 defined by the second member 108. The locking arm 144 is thus configured to engage with the first member to form a closed configuration, or to pivot (or rotate) relative to the second member 108. In other words, the locking arm 144 is configured to rotate relative to an axis defined by the second pivot member 148.

[0045] Return to reference Figure 6A Locking arm 144 defines a C-shaped (or U-shaped) member. The C-shaped member includes a central channel configured in a second closed configuration ( Figure 6B (As shown) a portion of the first member 104 is received in the second closed configuration. Therefore, the second closed configuration can also be called a locked configuration.

[0046] Insert 156 is configured to be positioned between first member 104 and second member 108. More specifically, insert 156 is configured to be confined (or sandwiched) between first member 104 and second member 108. Insert 156 is configured to be received by an offset space between first member 104 and second member 108. Insert 156 defines a plurality of third holes 160. The third holes 160 extend completely through insert 156. Thus, insert 156 defines a plurality of third holes 160. Each of the plurality of third holes 156 is configured to be perpendicularly aligned with a first hole of a plurality of first holes 128 and a second hole of a plurality of second holes 140. Thus, the plurality of third holes 156 are arranged in a configuration similar to both the first holes 128 and the second holes 140. In the illustrated embodiment, the plurality of third holes 160 are arranged in a grid pattern, and more specifically, in a 5×4 grid pattern. However, in other examples of the embodiments, the plurality of third holes 160 can be arranged in any suitable or desired pattern, such that each third hole 160 is perpendicularly aligned with the associated first hole 128 and second hole 140. Exemplary embodiments include 4×4, 5×4, 5×5, 5×8, 5×8, 6×6, 6×8, 8×8, and other iterations. Furthermore, the plurality of third holes 160 are shown as having a square shape. In other examples of the embodiments, the plurality of third holes 160 can have any suitable polygonal shape (e.g., circle, triangle, rectangle, pentagon, etc.). The shape of the third hole 160 is preferably the same as (or complementary to) the shape of the first hole 128 and the second hole 140. In the illustrated embodiment, the insert 156 is a silicone insert configured to be removable from module 100.

[0047] refer to Figure 6CIt should also be understood that, in the closed configuration, each of the first hole 128, the second hole 140, and the third hole 160 is aligned (or perpendicularly aligned). The aligned first hole 128, second hole 140, and third hole 160 define a channel that extends fully through the module 100. The first hole 128, second hole 140, and third hole 160 may also be referred to as the first recess 128, the second recess 140, and the third recess 160.

[0048] Manufacturing method

[0049] The tissue sample compartmentation module described in this article can be manufactured using standard manufacturing methods, including injection molding, 3D printing, casting, laser ablation, stamping, other methods, or combinations thereof.

[0050] The tissue sample compartmentation module may include materials such as plastics (polyethylene, polypropylene, polystyrene, polyoxymethylene, polytetrafluoroethylene, copolymers), metals (aluminum, titanium, stainless steel), glass, composite materials (graphite, carbon fiber, glass fiber), or combinations thereof. Inserts or gaskets may include waterproof materials such as silicone, closed-cell foam, open-cell foam, polytetrafluoroethylene, rubber, glass fiber, polyarylamide fiber, felt, cork, paper, or combinations thereof.

[0051] How to use

[0052] This document provides a method for compartmentalizing a tissue sample for labeling two or more analytes in the tissue sample. The method may include: placing a substrate containing the tissue sample onto a first member of a tissue sample compartmentalization module, wherein the tissue sample compartmentalization module includes a first member defining a first plurality of wells; and a second member defining a second plurality of wells, the second member being pivotally connected to the first member, the second member being configured to engage with the first member to form a closed configuration, or to pivot relative to the first member between an open configuration and a closed configuration, wherein in the closed configuration, the first plurality of wells and the second plurality of wells are aligned; aligning the tissue sample with the first member; placing an insert on the tissue sample such that a plurality of third wells defined by the insert are aligned with a plurality of first wells defined by the first member; engaging or pivoting the second member relative to the first member to clamp the insert and the tissue sample between the first member and the second member, and aligning the plurality of second wells with the plurality of third wells; and securing the first member and the second member together by a locking arm, thereby sealing the insert to the tissue sample. In one embodiment, aligning the tissue sample with the first member may further include making the plurality of third holes defined by the insert overlap with the plurality of first holes defined by the first member as much as possible with the tissue sample to create a plurality of sample compartments.

[0053] refer to Figures 7A to 7D This illustrates the procedure for using the tissue sample compartmentation module 100. See details. Figure 7AThe microscope slide 200 is shown as having multiple tissue sections 204a, 204b positioned thereon.

[0054] Next, in Figure 7B In this arrangement, multiple tissue sample compartmentation modules 100 are positioned to engage with a microscope slide 200. In the example shown, a pair of modules 100 are positioned to engage with the slide 200 such that one module 100 is associated with each tissue section 204a, 204b. Each module 100 is positioned in an open configuration, as shown below. Figure 6A As shown. A microscope slide 200 is positioned on the planar surface 124 of the first member 104 of each module 100. Protrusions 132, 136 of each member facilitate alignment of the slide 200 relative to the first member 104. Once the slide 200 is positioned on the planar surface 124 of the first member 104, the slide 200 can be moved laterally relative to the first member 104 to align the corresponding tissue sections 204a, 204b over the plurality of first apertures 128. An insert 156 is then positioned on the slide 200. The insert 156 is positioned over each corresponding tissue section 204a, 204b, wherein the plurality of third apertures 160 are perpendicularly aligned with the plurality of first apertures 128. The insert 156 can be manually positioned on the slide 200, or can be configured in response to a second member 108 opening from the first member 104 (see [link to relevant documentation]). Figure 6A From closed configuration (see) Figure 6B It is positioned on the glass slide 200 by the pivoting motion of the slide.

[0055] Now for reference Figure 7C Each module 100 is actuated into a closed configuration and locked. Once positioned on the slide, the second member 108 of each module 100 pivots relative to the first member 104 to align a plurality of second holes 140 with a plurality of third holes 160 of the insert 156. A locking arm 144 can then pivot relative to the second member 108 to engage with the first member 104, locking members 104, 108 together. It should be understood that the slide 200 and the insert 156 are sandwiched (or positioned) between members 104, 108. Furthermore, the second holes 140 and the third holes 160 cooperate to form a plurality of recesses that are spaced apart from the first hole 128 by the slide 200. The recesses provide access to tissue samples 204a, 204b on the slide 200. Thus, one or more materials can be introduced into each recess to interact with a portion of the tissue sample 204a, 204b positioned in the recess.

[0056] refer to Figure 7D Module 100 has been unlocked and disengaged from slide 200. Samples 204a and 204b on the slide are imaged using fluorescence microscopy and linearly unmixed as needed.

[0057] The methods described herein can be used for immunohistochemical assays, immunocytochemical assays, in-situ hybridization (ISH) assays, enzyme immunoassays (EIA), enzyme-linked immunoassays (ELISA), blotting (e.g., Western blotting, Northern blotting, Southern blotting), labeling within electrophoresis systems or on surfaces or arrays, or other routine assays known in the art.

[0058] For example, immunohistochemistry (IHC) provides a method for the in situ detection of targets in a sample or tissue specimen. See, for example, Mokry, Acta Medica 39(4): 129-140 (1996). IHC maintains the overall cellular integrity of the sample, thus allowing the detection of both the presence and location of the target of interest. Typically, the sample is fixed in formalin, embedded in paraffin, and sectioned for staining and subsequent light microscopy. Current IHC methods use direct labeling or labeling based on secondary antibodies or haptens. Examples of known IHC systems include, but are not limited to, EnVision™ (DakoCytomation), Powervision® (Immunovision, Springdale, Arizona), NBA™ kits (Zymed Laboratories Inc., South San Francisco, California), and HistoFine® (Nichirei Corp., Tokyo, Japan). The apparatus and methods disclosed herein can allow for signal enhancement, increased flexibility of IHC detection platforms, or combinations thereof.

[0059] Many types of samples are compatible with the apparatus and methods disclosed herein. Samples may include solids, such as tissue sections containing targets from organs. Samples may be derived from living material taken from any living organism, such as animals, mammals (e.g., humans), plants, fungi, archaea, or bacteria. Thus, samples may contain eukaryotic cells, archaea cells, or prokaryotic cells. Tissue samples may be derived from subjects selected from: humans, non-human primates, rats, mice, guinea pigs, rabbits, pigs, cattle, sheep, goats, horses, dogs, cats, fish, birds, reptiles, amphibians, insects, plants, fungi, bacteria, or combinations thereof.

[0060] Samples may include cellular samples, such as cell smears or colonies, or tissue samples derived from living organisms, such as tissue samples from organs. Samples may also include other naturally occurring samples (such as plant tissue samples) and synthetically derived samples (such as chemicals or industrial products and food).

[0061] Depending on the sample type and assay format, tissue or cell samples can be prepared using a variety of methods known to those skilled in the art. For example, tissue or cell samples can be fresh or preserved and can be, for example, rapidly frozen, smeared, dried, embedded, or fixed on a slide or other support. Samples can be prepared and stained using free-floating techniques. For example, before mounting tissue sections onto a slide for further processing and examination using the methods described herein, the tissue sections can be contacted with various reagents and wash buffers suspended or free-floating in a suitable container (e.g., a microcentrifuge tube).

[0062] Tissue sections can be mounted onto a glass slide or other substrate after incubation with an immunospecific reagent. The remainder of the staining process can then be performed after mounting. For example, for microscopy in IHC and ISH, the sample can be contained in a tissue section mounted on a suitable solid substrate. For photomicrographs, sections containing the sample can be mounted on a glass slide or other flat substrate to highlight certain morphological markers of the disease state or the detection of detectable targets through selective staining. The substrate can be a glass or plastic microscope slide (e.g., 26 × 75 × 1 mm or 1 × 3 × 0.04 inches).

[0063] For IHC, samples can be collected from an individual, fixed, and exposed to antibodies that specifically bind to, for example, a detectable target of interest. Sample processing steps may include, for example, antigen retrieval, exposure to a primary antibody, washing, exposure to a secondary antibody (optionally coupled to a suitable detectable label), washing, and exposure to a tertiary antibody linked to the detectable label. Washing steps can be performed using any suitable buffer or solvent, such as phosphate-buffered saline (PBS), Tris-buffered saline (TBS), or distilled water. Washing buffers may optionally contain detergents such as polyoxyethylene sorbitan monolaurate (TWEEN® 20) or octylphenoxypolyethoxyethanol (Nonidet P-40).

[0064] IHC samples may include, for example: formulations containing unfixed fresh tissue and / or cell or solution samples; fixed and embedded tissue samples, such as archived materials; and frozen tissue or cells. IHC staining procedures may include steps such as: cutting and trimming tissue, fixation, dehydration, paraffin infiltration, cutting into thin sections, mounting onto glass slides, baking, dewaxing, rehydration, antigen retrieval, blocking steps, application of primary antibody, washing, application of secondary antibody-enzyme conjugate, washing, application of a polymer-conjugated and enzyme-linked triple antibody, application of chromogenic substrate, washing, counterstaining, covering with a coverslip, and microscopic examination using a fluorescence microscope, followed by processing using imaging and unmixing software.

[0065] For example, ISH samples can be collected from individuals and fixed before being exposed to nucleic acid or nucleic acid analog probes on recognition units. The nucleic acids in the sample can first be denatured to expose target binding sites. Various counterstains or coatings can then be used to locate nucleic acid molecules or chromosomes within the ISH sample.

[0066] Tissue or cell samples can be fixed or embedded. For example, a fixative may be needed to preserve cells and tissues in a reproducible and realistic manner. Fixatives can also stabilize cells and tissues, thus protecting them from harsh handling and staining techniques. For example, samples containing tissue blocks, sections, or smears can be immersed in a fixative or dried (in the case of smears).

[0067] Many methods for fixing and embedding tissue samples are known, such as alcohol fixation and formalin fixation followed by paraffin embedding (FFPE). Any suitable fixative can be used. Examples include ethanol, acetic acid, picric acid, 2-propanol, 3,3′-diaminobenzidine tetrahydrochloride dihydrate, acetoin (monomer mixtures) and dimers, acrolein, crotonaldehyde (cis + trans), formaldehyde, glutaraldehyde, glyoxal, potassium dichromate, potassium permanganate, osmium tetroxide, paraformaldehyde, mercuric chloride, toluene-2,4-diisocyanate, trichloroacetic acid, and tungstic acid. Other examples include formalin (formaldehyde aqueous solution), neutral buffered formalin, glutaraldehyde, carbodiimide, imine esters, benzoquinone, osmium tetroxide, and osmium tetroxide. Fresh biopsy samples, cytological preparations (including touch preparations and blood smears), frozen sections, and tissues for IHC analysis can be fixed in organic solvents, including ethanol, acetic acid, methanol, and / or acetone.

[0068] Pretreatment of samples can help improve the reactivity or accessibility of detectable targets and reduce nonspecific interactions. For example, if the target is an antigen, a process called “antigen retrieval” can be used (and it is also known in the art as target retrieval, epitope retrieval, target unblinding, or antigen unblinding). See, for example, Shi et al., J. Histochem. Cytochem. 45(3): 327-343 (1997). Antigen retrieval encompasses a variety of methods, including enzymatic digestion using proteases such as proteases, streptomycin, pepsin, papain, trypsin, or neuraminidase. Heating, such as heat-induced epitope retrieval or HIER, can also be used. Heating can involve microwave radiation, water bath, steam oven, conventional oven, autoclave, or pressure cooker in a pH-stabilized buffer. The buffer typically contains ethylenediaminetetraacetic acid (EDTA), ethylene glycol diethyl ether diaminetetraacetic acid (EGTA), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), citrate, urea, glycine hydrochloride (glycin-HCl), or boric acid. Detergents can be added to HIER buffers to enhance epitope retrieval or to dilution media and / or rinse buffers to reduce nonspecific binding. Combinations of different antigen retrieval methods can be used. Antigen retrieval buffers can be aqueous but may also contain other solvents, including those with boiling points above water, such as glycerol. This allows tissue treatment at temperatures above 100°C under standard pressure.

[0069] Before or during reagent incubation, the signal-to-noise ratio can be increased by various physical methods, including applying vacuum, ultrasound, or freezing and thawing tissue samples.

[0070] Treatments can be employed to reduce nonspecific binding. For example, carrier proteins, carrier nucleic acid molecules, salts, or detergents can reduce or prevent nonspecific binding. In some embodiments, nonspecific binding sites can be blocked with inert proteins (such as HSA, BSA, ovalbumin), fetal bovine serum or other sera, or with detergents (such as polyoxyethylene sorbitol monolaurate (TWEEN® 20), octylphenoxypolyethoxyethanol (Nonidet P-40), tert-octylphenol polyoxyethylene ether (TRITON™ X-100), triterpenoid saponins (saponins), nonionic polyoxyethylene surfactants (BRIJ®-35), or nonionic triblock copolymers (PLURONICS®)). Alternatively, for recognition events between the target and the affinity molecule, nonspecific binding sites can be blocked with unlabeled competitors. For example, in the case of nucleic acid interactions, nonspecific binding can be reduced by adding unlabeled competing nucleic acids or nucleic acid analogs, such as digested total human DNA or unlabeled versions of affinity molecules. In addition, repetitive sequences can be blocked, for example, by using nucleic acids or nucleic acid analogs that specifically recognize those sequences, or sequences derived from total DNA preparations. Salt, buffer, and temperature conditions can also be adjusted to reduce nonspecific binding.

[0071] For example, by using antibodies derived from different species, cross-reactivity between different components of the detection method can be avoided. Furthermore, combinations of secondary antibodies, such as those targeting primary antibodies and haptens, can be used to avoid unwanted cross-reactivity. Endogenous biotin-binding sites or endogenous enzyme activities (e.g., phosphatase, catalase, or peroxidase) can be removed as a step in the staining procedure. Endogenous biotin and peroxidase activity can be removed by treatment with peroxides, while endogenous phosphatase activity can be removed by treatment with levamisole. Heating can destroy endogenous phosphatase and esterase activities.

[0072] Methods for culturing cells on a substrate

[0073] This article describes a method for culturing cells on a substrate such as a glass microscope slide. The method may include: treating the substrate with a substance that promotes cell adhesion; placing the treated substrate onto a first member of a tissue sample compartmentation module, wherein the tissue sample compartmentation module includes a first member defining a first plurality of wells; and a second member defining a second plurality of wells, the second member being pivotally connected to the first member, the second member being configured to engage with the first member to form a closed configuration, or to pivot relative to the first member between an open configuration and a closed configuration, wherein in the closed configuration, the first plurality of wells and the second plurality of wells are aligned; aligning the treated substrate onto the first member; and placing an insert onto the treated substrate to allow the insert to be inserted into the cell compartmentation module. The insert, defined by a plurality of third wells, is aligned with a plurality of first wells defined by a first member; a second member is engaged or pivoted relative to the first member to clamp the insert and the treated substrate between the first and second members, and to align the plurality of second wells with the plurality of third wells; the first and second members are fastened together by locking arms, thereby sealing the insert to the treated substrate; a solution containing cells and culture medium is dispensed into one or more of the aligned second and third wells; the wells are sealed; and the tissue sample compartmentation module / treated substrate containing cell culture is incubated under conditions that promote cell culture growth. After incubation for a period of time, the cells can be imaged or fixed and stained with immunohistochemical reagents, as described herein. The sample can be preserved by disassembling the tissue sample compartmentation module and covering the cells with mounting media and coverslips.

[0074] Methods for performing immunohistochemistry

[0075] This document describes a method for labeling one or more analytes in a tissue sample described herein. The method may include: placing a substrate containing the tissue sample onto a first member of a tissue sample compartmentalization module, wherein the tissue sample compartmentalization module includes a first member defining a first plurality of wells; and a second member defining a second plurality of wells, the second member being pivotally connected to the first member, the second member being configured to engage with the first member to form a closed configuration, or to pivot relative to the first member between an open configuration and a closed configuration, wherein in the closed configuration, the first plurality of wells and the second plurality of wells are aligned; aligning the tissue sample with the first member; placing an insert on the tissue sample such that a plurality of third wells defined by the insert are aligned with a plurality of first wells defined by the first member; engaging or pivoting the second member relative to the first member to clamp the insert and the tissue sample between the first member and the second member, and aligning the plurality of second wells with the plurality of third wells; securing the first member and the second member together by a locking arm to seal the insert to the tissue sample; and incubating a solution containing affinity molecules in the aligned second and third wells, wherein the affinity molecules label analytes in the tissue sample.

[0076] This method may also include labeling two or more different analytes. Each aligned second and third well of the tissue sample compartmentation module described herein may include a solution containing different affinity molecules for each analyte. The method described herein may further include simultaneous multiplexing of two or more targets, wherein each of the two or more target molecules is individually bound by an affinity molecule. Simultaneous multiplexing of 10 or more targets is possible, wherein each of the 10 or more target molecules is individually bound by an affinity molecule. Simultaneous multiplexing of 100 or more targets is possible, wherein each of the 100 or more target molecules is individually bound by an affinity molecule.

[0077] This document also describes a method for analyzing tissue samples for a variety of analytes. The method may include: placing a substrate containing the tissue sample onto a first member of a tissue sample compartmentation module, wherein the tissue sample compartmentation module includes a first member defining a first plurality of wells; and a second member defining a second plurality of wells, the second member being pivotally connected to the first member, the second member being configured to engage with the first member to form a closed configuration, or to pivot relative to the first member between an open configuration and a closed configuration, wherein in the closed configuration, the first plurality of wells and the second plurality of wells are aligned; aligning the tissue sample with the first member; and placing an insert onto the tissue sample to allow the plurality of wells defined by the insert to... The third well is aligned with a plurality of first wells defined by the first member; the second member is engaged or pivoted relative to the first member to clamp the insert and tissue sample between the first and second members, and the plurality of second wells are aligned with a plurality of third wells; the first and second members are fastened together by locking arms, thereby sealing the insert to the tissue sample; a solution containing affinity molecules is incubated in the aligned second and third wells, wherein the affinity molecules label analytes in the tissue sample; and signals from each affinity molecule bound to the plurality of analytes are detected, thereby detecting the presence or amount of each analyte in the plurality of analytes.

[0078] The tissue samples described herein may be from subjects selected from the following: humans, non-human primates, rats, mice, guinea pigs, rabbits, pigs, cattle, sheep, goats, horses, dogs, cats, fish, birds, reptiles, amphibians, insects, plants, fungi, bacteria, or combinations thereof.

[0079] The one or more analytes described herein may include one or more of metabolites, proteins, nucleic acids, carbohydrates, or lipids.

[0080] The affinity molecules described herein can be antibodies, portions of antibodies, antibody-like molecules, ligand receptors, ligands of receptors, members of a coupling pair, aptamers, or antigens. Affinity molecules can include primary antibodies. Affinity molecules can include secondary antibodies. Affinity molecules can be conjugated to fluorescent molecules, conjugated to enzymes, or bound to another affinity molecule conjugated to a fluorescent molecule. In the methods described herein, the method may include the use of more than one affinity molecule, for example, two affinity molecules, three affinity molecules, four affinity molecules, five affinity molecules, six affinity molecules, seven affinity molecules, eight affinity molecules, nine affinity molecules, or ten affinity molecules. More than ten affinity molecules may be used in any of the methods described herein.

[0081] The affinity molecules described herein can be detected by signals from detectable tags associated with the affinity molecules, which may be directly or indirectly associated with the affinity molecules. Detectable tags used herein may include fluorophores, polymer particles, metal particles, haptens, enzymes, luminescent labels, radioactive labels, etc.

[0082] Examples of fluorophores include fluorescein or its derivatives, such as fluorescein-5-isothiocyanate (FITC), 5-(and 6)-carboxyfluorescein, 5- or 6-carboxyfluorescein, 6-(fluorescein)-5-(and 6)-carboxyhexanoic acid, fluorescein isothiocyanate, rhodamine or its derivatives, such as tetramethylrhodamine and tetramethylrhodamine-5-(and-6)-isothiocyanate (TRITC). Other examples of fluorophores that can conjugate with affinity molecules include, but are not limited to: coumarin dyes, such as (diethylamino)coumarin or 7-amino-4-methylcoumarin-3-acetic acid succinimidyl ester (AMCA); sulforhodamine 101 sulfonyl chloride (TexasRed™ or TexasRed™ sulfonyl chloride; 5-(and-6)-carboxy-X-rhodamine, succinimidylester, also known as 5-(and-6)-carboxy-X-rhodamine, succinimidylester (CXR); lissamine or lissamine derivatives, such as lissaminerhodamine B sulfonyl chloride (LisR); 5-(and-6)-carboxyfluorescein, succinimidyl ester (hereinafter referred to as CFI); fluorescein-5-isothiocyanate (hereinafter referred to as FITC); 7-diethylaminocoumarin-3-carboxylic acid, succinimidyl ester (hereinafter referred to as DECCA); 5-(and-6)-carboxytetramethylrhodamine, succinimidyl ester (hereinafter referred to as CTMR); 7-hydroxycoumarin-3-carboxylic acid, succinimidyl ester (hereinafter referred to as HCCA); 6-fluorescein-5-(and-6)-carboxamidolhexanoic acid (hereinafter referred to as FCHA);N-(4,4-difluoro-5,7-dimethyl-4-boron-3a,4a-diaza-3-indophenylpropionic acid succinimidyl ester; also known as 5,7-dimethylBODIPY™ propionic acid, succinimidyl ester (DMBP); "activated fluorescein derivative" (FAP), available from Molecular Probes, Inc.; eosin-5-isothiocyanate (EITC); erythrosin-5-isothiocyanate (ErITC); and Cascade™ Blue acetylated azide. acetylazide (hereinafter referred to as CBAA) (an O-acetylazide derivative of 1-hydroxy-3,6,8-pyrene trisulfonic acid). Other potential fluorophores useful in this document include, but are not limited to: fluorescent proteins, such as green fluorescent protein (hereinafter referred to as GFP) and its analogues or derivatives; fluorescent amino acids, such as tyrosine and tryptophan and their analogues; fluorescent nucleosides; and other fluorescent molecules, such as Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, infrared dyes, Dyomics dyes, phycoerythrin, Oregon Green 488, Pacific Blue, Rhodamine Green, and Alexa dyes. Other examples of fluorescent labels that may be used in this document include conjugates of phycoerythrin, and inorganic fluorescent labels, such as particles based on semiconductor materials (e.g., coated CdSe nanocrystals). Several of the above-mentioned fluorophores, as well as others, may be obtained from sources such as Molecular Probes, Inc. (Eugene, Oreg.), Pierce Chemical Co. (Rockford, Ill.), and Sigma-Aldrich Co. (St. Louis, Acquired through a commercial purchase by a company in Mo.

[0083] Examples of polymer particle labeling include, but are not limited to, microparticles, microspheres or latex particles of polystyrene, polymethyl methacrylate (PMMA) or silica (which may contain embedded fluorescent dyes) or polymer micelles or vesicles containing dyes, enzymes or substrates.

[0084] Examples of metal particles include, but are not limited to, gold particles and coated gold particles, which can be converted by silver dye.

[0085] Examples of haptens include, but are not limited to, fluorophores, myc, nitrotyrosine, biotin, avidin, streptavidin, 2,4-dinitrophenyl, isohydroxydigitotoxin, bromodeoxyuridine, sulfonates, acetaminophen, trinitrophenol mercury, and estradiol.

[0086] Examples of enzymes include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, β-N-acetylglucosamine glycosidase, β-glucuronidase, invertase, xanthine oxidase, firefly luciferase, and glucose oxidase (GO).

[0087] Examples of commonly used substrates for horseradish peroxidase (HRP) include, but are not limited to, Alexa Fluor™ 350, Alexa Fluor™ 488, Alexa Fluor™ 546, Alexa Fluor™ 555, Alexa Fluor™ 568, Alexa Fluor™ 594 and Alexa Fluor™ 647 tyrosamide reagents or biotin-XX tyrosamide reagents (Thermo Fisher Scientific), 3,3′-diaminobenzidine (DAB), nickel-enhanced diaminobenzidine, 3-amino-9-ethylcarbazole (AEC), Benzidine dihydrochloride (BDHC), Hanker-Yates reagent (HYR), and Indocyanine. blue (hereinafter referred to as IB), tetramethylbenzidine (hereinafter referred to as TMB), 4-chloro-1-naphtol (hereinafter referred to as CN), α-naphtolpyronin (hereinafter referred to as α-NP), o-dianisidine (hereinafter referred to as OD), 5-bromo-4-chloro-3-indolylphosphate (hereinafter referred to as BCIP), Nitroblue tetrazolium (hereinafter referred to as NBT), 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (hereinafter referred to as INT), tetranitroblue tetrazolium (hereinafter referred to as INT). Tetrazolium (hereinafter referred to as TNBT) and 5-bromo-4-chloro-3-indoxyl-β-d-galactoside / ferro-ferricyanide (hereinafter referred to as BCIG / FF).

[0088] Examples of commonly used substrates for alkaline phosphatase include, but are not limited to, Naphthol-AS-B1-phosphate / fast red TR (NABP / FR), Naphthol-AS-MX-phosphate / fast red TR (NAMP / FR), Naphthol-AS-B1-phosphate / fast red TR (NABP / FR), Naphthol-AS-MX-phosphate / fast red TR (NAMP / FR), Naphthol-AS-B1-phosphate / new fuschin (NABP / NF), and bromochloroindolyl phosphate / nitroblue tetrazolium. tetrazolium (hereinafter referred to as BCIP / NBT) and 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (hereinafter referred to as BCIG).

[0089] Examples of luminescent labels include, but are not limited to, luminol, isoluminol, acridine esters, 1,2-dioxane, and pyridopyridazine. Examples of electrochemiluminescent labels include, but are not limited to, ruthenium derivatives.

[0090] Examples of radiolabeled materials include, but are not limited to, radioactive isotopes of iodine, cobalt, selenium, hydrogen, carbon, sulfur, and phosphorus.

[0091] Labeled analytes can be detected by a variety of methods, including, for example, by reflectance, transmittance, light scattering, optical rotation, fluorescence, or combinations thereof; radioactive labels can be detected by film, scintillation counting, or phosphorescence imaging. See, for example, Larsson, Immunocytochemistry: Theory and Practice, CRC Press, Boca Raton, FL 1988; Methods in Molecular Biology 80, J. Pound (ed.), Humana Press, Totowa, NJ (1998). More than one detectable label can be used. When more than one color label is used, the different colors can have different, distinguishable colors. Two colors can be detected simultaneously, such as by signal fusion or juxtaposition, signal enhancement or quenching, or detection of multiple colors in the sample. The exact selection of a detectable label or combination of detectable labels can be based on personal preference, combined with limitations of sample type, sample preparation method, detection method and equipment, and optional contrast labels used in the sample.

[0092] The apparatus and methods disclosed herein are applicable to a wide variety of targets. Any target that can be recognized by a suitable affinity molecule is compatible with the apparatus and methods described herein. This recognition can be direct or indirect, via another affinity molecule, such as at least one primary, secondary, or higher affinity molecule.

[0093] Targets or analytes may include proteins, such as glycoproteins or lipoproteins, phosphoproteins, methylated proteins or protein fragments, peptides or polypeptides. Targets or analytes may contain nucleic acid fragments or nucleic acid analog fragments.

[0094] Targets or analytes may comprise lipids; glycolipids; carbohydrates; polysaccharides; salts; ions; or one or more of various other organic and inorganic substances. Targets or analytes may be expressed on the surface of the sample, such as on membranes or interfaces. Alternatively, targets or analytes may be contained within the sample. For example, in the case of cell samples, internal targets or analytes may include those located within the cell membrane, periplasmic space, cytoplasm, or nucleus, or within intracellular compartments or organelles.

[0095] Targets or analytes may also include viral particles or portions thereof, such as nucleic acids or proteins. Viral particles may be free viral particles, i.e., not associated with any other molecules, or they may be associated with any of the samples described above. Targets or analytes may be antigens or antibodies.

[0096] The approximate amount of a target in a sample can be determined. For example, control targets and experimental targets can be measured within the sample. For instance, in the case of nucleic acid targets, chromosome smearing or counterstaining can be used. For example, if the target is a locus on a large nucleic acid fragment (such as a plasmid or chromosome), the intensity of a contrast marker at a neutral locus on the plasmid or chromosome can be compared to the intensity of the target locus. The intensity of a marker from the sample can also be compared to the intensity of a known standard or control sample. For example, estimating the amount of detectable targets in a sample is helpful in various diagnostic tests, and this estimate can be used to plan the treatment process for a suspected disease or condition. Several commercial density measurement software programs and related instruments are available for quantifying the intensity of stained targets in a sample, such as those available from Fuji Film, Applied Biosystems, and Molecular Dynamics.

[0097] Reagent test kit

[0098] This document also describes a kit containing one or more tissue sample compartmentation modules as described herein. For example, the kit may contain modules with various compartment arrays, such as 4×4 (16 compartments), 5×4 (20 compartments), 6×6 (36 compartments), 8×8 (64 compartments), or fewer or more wells / compartments to accommodate tissue samples of different sizes. The kit may optionally contain one or more affinity molecules; one or more signal generation reagents; and optionally, one or more reagents for performing the methods described herein, such as: deparaffinizing reagents for removing paraffin from FFPE slides, enzyme activation buffers (e.g., Tris buffer + peroxide cofactor for activating HRP), antigen retrieval, sample dilution, reagent dilution, nonspecific binding blocking, or endogenous enzyme activity blocking. The kit may optionally include one or more containers, packaging, instructions for use, MSDS sheets, or reference or control tissue samples and / or targets.

[0099] It will be apparent to those skilled in the art that suitable modifications and adaptations can be made to the compositions, formulations, methods, processes, and applications described herein without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and not intended to limit the scope of any specified embodiment. Each of the various embodiments, aspects, and options disclosed herein can be combined in any variation or iterative manner. The scope of the compositions, formulations, methods, and processes described herein includes all actual or potential combinations of embodiments, aspects, options, examples, and preferred embodiments described herein. Exemplary compositions and formulations described herein may omit any component, substitute for any component disclosed herein, or include any component disclosed elsewhere herein. In any composition or formulation disclosed herein, the mass ratio of any component to the mass of any other component in the formulation or to the total mass of other components in the formulation is deemed to be explicitly disclosed. If the meaning of any term in any patent or publication incorporated herein by reference conflicts with the meaning of the term used in this disclosure, the meaning of the term or phrase in this disclosure shall prevail. Furthermore, the specification discloses and describes only exemplary embodiments. All patents and publications referenced herein are incorporated herein by reference for their specific teaching.

[0100] Example

[0101] Example 1

[0102] Standard tissue immunohistochemistry.

[0103] Immunohistochemical (IHC) staining is used to detect specific antigens in tissues. Typically, tissue samples are fixed in formalin to preserve tissue integrity. Paraffin-embedded tissue sections must be dewaxed and then rehydrated before primary antibody application. Frozen tissue sections embedded in optimal cutting temperature (OCT) compounds must be rehydrated before primary antibody application. Enzyme-conjugated secondary antibodies are then applied, and specific staining is observed after the addition of an enzyme-specific substrate. Occasionally, when weak or no staining is observed, antigen exposure or signal enhancement can be achieved through the methods described herein, such as enzymatic digestion or microwave antigen retrieval.

[0104] Permeabilize sample sections on a substrate such as a glass slide at room temperature (RT, approximately 20°C to 25°C) for about 30 minutes. Then, before reacting with the primary antibody, incubate the permeabilized sample with a blocking agent (such as approximately 2% to 10% normal serum from the same host species as the secondary antibody) at RT for about 60 minutes. The blocking agent reduces background staining. Determine the optimal dilution and incubation time for each primary antibody before use. Drain the slides and remove excess liquid around the sample sections on each slide. Dilute the primary antibody or negative control reagent to its optimal dilution in the diluent. The primary antibody can be directly conjugated to the fluorophore. The diluent alone can be used as a negative control. A positive control (i.e., tissue known to contain the target antigen) will also be included. Apply an appropriate amount of the diluted primary antibody solution to a suitable slide, cover the tissue section, and incubate in a humidified chamber at RT for at least 60 minutes or an appropriate time, or overnight at 4°C. After incubation, gently rinse the slide with PBS, and then incubate the slide in a PBS wash bath for about 1 to 5 minutes.

[0105] If the primary antibody is not conjugated to the fluorophore, a secondary antibody or enzyme conjugated to the fluorophore will be used. Dilute the secondary antibody to its optimal dilution in diluent. Apply an appropriate amount of the secondary antibody solution to a suitable glass slide, cover the tissue section, and incubate at room temperature for at least 30 minutes or as appropriate. Store the sample protected from light. After incubation, wash the slide three times in washing buffer for 5 to 15 minutes each time.

[0106] If the secondary antibody is not conjugated to a fluorophore but to an enzyme (such as horseradish peroxidase "HRP" or alkaline phosphatase "AP"), a tertiary detection substrate, such as fluorescent tyrosine, will be used. Fluorescent tyrosine is diluted to its optimal concentration in a diluent consisting of HRP reaction buffer and the desired cofactor (such as hydrogen peroxide). An appropriate amount of reactive tyrosine is applied to a suitable slide, covering the tissue section, and incubated at RT for 2 to 10 minutes. Samples are stored protected from light. After incubation, HRP enzyme activity can be quenched using a reaction termination reagent, and the slides are washed three times in wash buffer for 5 to 15 minutes each time. Slides can be mounted with an anti-fading mounting medium. The slides are observed using a fluorescence microscope, and the fluorescence data are analyzed and unmixed using software.

[0107] Example 2

[0108] Tissue immunohistochemistry using the tissue sample compartmentation module.

[0109] Typically, tissue samples are fixed in formalin to preserve tissue integrity. If tissue sections are embedded in paraffin, they must be dewaxed and then rehydrated before primary antibody application. If tissue sections are frozen and embedded in OCT, they must be rehydrated before primary antibody application. Antigen exposure or signal enhancement can be achieved through methods described herein, such as enzymatic digestion or microwave antigen retrieval.

[0110] Tissue sample sections on a substrate such as a glass slide (“tissue sample / substrate”) are permeated under RT for approximately 30 minutes. Prior to primary antibody reaction, the permeated tissue sample is incubated under RT for approximately 60 minutes with an inhibitor (such as approximately 2% to 10% BSA or normal serum from the same host species as the secondary antibody). The inhibitor reduces background staining.

[0111] After dewaxing / antigen retrieval, permeabilization, and blocking, the sample is placed in the compartmentalized module, and the module is removed for washing after antibody staining. Removing liquid from the wells by pipetting can damage the tissue, so all steps that can be performed on the entire sample should be done without the module. However, if a secondary antibody is to be used and the wells need to be washed separately, the entire microscope slide can be immersed in the washing solution with the device in place.

[0112] A tissue sample / substrate is placed onto a first member of the tissue sample compartmentation module described herein, and the tissue sample / substrate is aligned with the first member. In one aspect, a plurality of first holes defined by the first member are aligned to overlap the tissue sample with as many holes as possible to provide a plurality of sample compartments. An insert is placed or positioned on the tissue sample / substrate such that a plurality of third holes defined by the insert are aligned with a plurality of first holes defined by the first member. A second member is then positioned on the tissue sample and aligned with the first member, clamping the substrate and tissue sample between the first and second members. The second member pivots relative to the first member to clamp the insert and tissue sample / substrate between the first and second members and to align the plurality of second holes with the plurality of third holes. The first and second members are fastened together by locking arms, thereby sealing the insert to the tissue sample / substrate and forming a plurality of individual sample compartments.

[0113] Determine the optimal dilution and incubation time for each primary antibody before use. Empty the slide and remove excess liquid. Dilute the primary antibody or negative control reagent to its optimal dilution in the diluent. The primary antibody can be directly conjugated to the fluorophore. The diluent alone can be used as a negative control. Positive controls (i.e., tissues known to contain the target antigen) may also be included. Apply an appropriate amount of the primary antibody solution to each sample compartment, covering the tissue section fragment in each sample compartment, and incubate in a humidified chamber at 37°C for at least 60 minutes or as appropriate. After incubation, gently rinse the sample compartments with PBS, and the sample compartments can be incubated in a PBS wash bath for approximately 1 to approximately 5 minutes.

[0114] If the primary antibody is not conjugated to the fluorophore, use a secondary antibody that is conjugated to the fluorophore. Dilute the secondary antibody to its optimal dilution in diluent. Apply an appropriate amount of the secondary antibody solution to the appropriate sample compartment, covering the tissue section fragment in each sample compartment, and incubate at room temperature for at least 30 minutes or as appropriate. Store the samples protected from light. After incubation, wash the sample compartments three times in washing buffer for 5 to 15 minutes each time.

[0115] If the secondary antibody is not conjugated to a fluorophore but to an enzyme (such as horseradish peroxidase (HRP)), a tertiary detection substrate, such as fluorescent tyrosine, will be used. Fluorescent tyrosine is diluted to its optimal concentration in a diluent consisting of HRP reaction buffer and the desired cofactor (such as hydrogen peroxide). An appropriate amount of reactive tyrosine is applied to a suitable slide, covering the tissue section, and incubated at RT for 2 to 10 minutes. The sample is stored protected from light. After incubation, HRP enzyme activity can be quenched using a reaction termination reagent, and the slide is washed three times in washing buffer for 5 to 15 minutes each time. The tissue sample compartmentation module can be removed, and the slide is mounted with an anti-fading mounting medium. The slide is then observed using a fluorescence microscope.

[0116] Example 3

[0117] Fluorophore overflow experiment using tissue sample compartmentation module.

[0118] The aim of this experiment was to demonstrate that the design of the 3D-printed tissue sample compartmentation module prevents fluorophores from spilling from one sub-compartment to another, and that tissue separated by sub-compartments can be stained independently of surrounding areas. Each sub-compartment was stained with only one primary antibody conjugated to the fluorophore. The normalized average spectral intensity of the compartmented samples was compared with the spectral profiles extracted from control tissue samples stained without compartmentation and without spillage.

[0119] Three normal human tonsil formalin-fixed paraffin-embedded (FFPE) tissue samples (Zyagen) were dewaxed using the Biogenex EZ-AR2 Elegance solution in the Biogenex EZ-retriever microwave antigen retrieval system (Biogenex) with two thermal cycles: 95°C for 5 minutes and 107°C for 5 minutes. The samples were permeabilized in phosphate-buffered saline (PBS) with 0.1% t-octylphenoxypolyethoxyethanol (Triton™ X-100) at RT for 30 minutes to improve antibody penetration into the tissue. Permeabilized tissue was blocked at RT for 60 minutes using PBS containing 3% bovine serum albumin (BSA) to prevent nonspecific binding. Nonspecific binding of dye-conjugated antibodies was also reduced by treating the samples at RT for 30 minutes with Image-IT FX signal enhancer (Invitrogen).

[0120] Samples were stained with primary antibodies conjugated to fluorophores. Two control samples were stained with monoclonal antibody-fluorophore conjugates (CD20 monoclonal antibody (L26), Alexa Fluor™ 488, eBioscience™ (Invitrogen) and vimentin monoclonal antibody (V9), Alexa Fluor™ 555 (Invitrogen)). A tissue sample was partitioned into a subcompartment using a tissue compartmentation module equipped with silicone pads (i.e., the device described herein). The subcompartment was stained with the same antibody conjugates used to stain the control samples.

[0121] Images were acquired using 25 spectral channels for each sample. Characteristic dye curves were obtained from all images by averaging the intensity of the entire image for each acquired channel.

[0122] The normalized average spectral intensities collected from the compartmentalized sample and the control sample were almost identical. See also Figures 8A to 8B These experiments demonstrate that the tissue compartmentation module enables independent staining within subcompartments, and that fluorophores from subcompartments do not exhibit overlapping spectral profiles.

[0123] Example 4

[0124] Cell-based applications of tissue compartmentalization modules.

[0125] If using standard uncoated glass microscope slides, pretreatment is required to allow cell adhesion. Since cells do not readily adhere to glass, the slide surface must first be coated with a substance that promotes cell adhesion, such as surface-polarized polyamino acids (e.g., poly-L-lysine, poly-D-lysine, polyornithine), non-specific protein coatings (e.g., gelatin), specific extracellular matrix components (e.g., purified I and IV collagen, fibronectin, laminin, fibronectin, osteopontin), specific partial sequences of extracellular matrix proteins (e.g., RGD-Motivs, fibronectin domains), or specific mixtures of extracellular matrix proteins (e.g., soluble basement membrane preparations, Corning Matrigel®; Thermo Fisher Scientific Geltrex™). The entire slide surface can be coated, or if the modules have been assembled onto the slide prior to coating, only the area covered by the module's well array can be coated. Once the slide surface has been modified for cell adhesion, the modules are assembled onto the slide (if not already assembled), and the cell suspension is transferred into the wells of a cell-specific culture medium. The number of cells per well depends on the experiment, the cell type used, and the well size. The modules are sealed to prevent contamination but allow gas exchange, and the slide-module complex is placed in an incubator to allow cell adhesion. Incubation time and environmental conditions depend on the cell type used. After the cells have adhered and reached the desired confluence, the user can proceed with the experimental protocol. The number of individual experimental environments per slide depends on the number of wells per module and the number of modules per slide. For example, by using two 5×4 tissue compartmentation modules, the user can obtain 40 unique experimental environments on a single slide. These experiments can be, for example, drug therapy, gene modification, toxicology assays, or cell differentiation assays. After exposing the cells to the experimental environments, they can be imaged in vivo using a microscope or further processed, for example, by fixation and staining with immunohistochemistry. Samples can be preserved by disassembling the modules and covering the cells with a coverslip and mounting medium. The preserved coverslip can be further examined using high-magnification microscopy.

Claims

1. A sample compartmentation module, the sample compartmentation module comprising: A first component, the first component defining a plurality of first holes; as well as A second component, defining a plurality of second holes, is pivotally connected to the first component. The second member is configured to engage with the first member to form a closed configuration, or to pivot relative to the first member between an open configuration and the closed configuration, wherein in the closed configuration, the plurality of first holes and the plurality of second holes are aligned.

2. The sample compartmentation module according to claim 1, characterized in that, The sample compartmentation module further includes a locking arm coupled to the second member, the locking arm being configured to rotate relative to the second member, wherein in the closed configuration, the locking arm is configured to engage a portion of the first member to secure the first member and the second member together.

3. The sample compartmentation module according to claim 2, characterized in that, The locking arm defines a central channel, wherein in the closed configuration, the central channel of the locking arm is configured to receive a portion of the first member.

4. The sample compartmentation module according to claim 1, characterized in that, The sample compartmentation module further includes an insert defining a plurality of third holes, wherein in the closed configuration, the insert is positioned between the first member and the second member, and the plurality of third holes are aligned with the plurality of first holes and the plurality of second holes.

5. A method for labeling one or more analytes in a tissue sample, the method comprising: a. Place the substrate containing the tissue sample onto the first component of the sample compartmentation module according to claim 1; b. Align the tissue sample with the first component; c. Place the insert on the tissue sample such that the plurality of third holes defined by the insert are aligned with the plurality of first holes defined by the first member; d Engage or pivot the second member relative to the first member to clamp the insert and the tissue sample between the first member and the second member, and align the plurality of second holes with the plurality of third holes; e secures the first and second components together using a locking arm, thereby sealing the insert to the tissue sample; as well as f. Incubate solutions containing affinity molecules in aligned second and third wells, wherein the affinity molecules label the analytes in the tissue sample.

6. The method according to claim 5, characterized in that, The samples were taken from subjects selected from the following: humans, non-human primates, rats, mice, guinea pigs, rabbits, pigs, cattle, sheep, goats, horses, dogs, cats, fish, birds, reptiles, amphibians, insects, plants, fungi, bacteria, or combinations thereof.

7. The method according to claim 5, characterized in that, The analytes include one or more of metabolites, proteins, nucleic acids, carbohydrates, or lipids.

8. The method according to claim 5, characterized in that, The method further includes labeling two or more different analytes, wherein each aligned second and third well comprises a solution containing a different affinity molecule for each analyte.

9. The method according to claim 5, characterized in that, The affinity molecule is an antibody, a part of an antibody, an antibody-like molecule, a ligand receptor, a ligand of a receptor, a member of a coupling pair, an aptamer, or an antigen.

10. The method according to claim 5, characterized in that, The affinity molecule is conjugated to a fluorescent molecule, conjugated to an enzyme, bound to another affinity molecule conjugated to a fluorescent molecule, or bound to another affinity molecule conjugated to an enzyme.

11. The method according to claim 5, characterized in that, The substrate is a microscope slide.

12. A method for dividing a sample into compartments for labeling two or more analytes in a tissue sample, the method comprising: a. Place the substrate containing the tissue sample onto the first component of the sample compartmentation module according to claim 1; b. Align the tissue sample with the first component; c. Place the insert on the tissue sample such that the plurality of third holes defined by the insert are aligned with the plurality of first holes defined by the first member; d Engage or pivot the second member relative to the first member to clamp the insert and the tissue sample between the first member and the second member, and align the plurality of second holes with the plurality of third holes; as well as e secures the first and second components together via a locking arm, thereby sealing the insert to the tissue sample.

13. The method according to claim 12, characterized in that, The sample is a tissue sample from a subject selected from the following: human, non-human primate, rat, mouse, guinea pig, rabbit, pig, cow, sheep, goat, horse, dog, cat, fish, bird, reptile, amphibian, insect, plant, fungus, bacteria, or a combination thereof.

14. The method according to claim 12, characterized in that, The substrate is a microscope slide.

15. The method according to claim 12, characterized in that, Step c further includes making the plurality of first pores defined by the first member overlap with the tissue sample as much as possible.

16. A method for analyzing tissue samples using multiple analytes, the method comprising: a. Place the substrate containing the tissue sample onto the first component of the sample compartmentation module according to claim 1; b. Align the tissue sample with the first component; c. Place the insert on the tissue sample such that the plurality of third holes defined by the insert are aligned with the plurality of first holes defined by the first member; d Engage or pivot the second member relative to the first member to clamp the insert and the tissue sample between the first member and the second member, and align the plurality of second holes with the plurality of third holes; e secures the first and second components together using a locking arm, thereby sealing the insert to the tissue sample; as well as f. Incubate solutions containing affinity molecules in aligned second and third wells, wherein the affinity molecules label the analytes in the tissue sample; g detects the signal from each affinity molecule that binds to the plurality of analytes, thereby detecting the presence or amount of each of the plurality of analytes.

17. The method according to claim 16, characterized in that, Detection is performed using a light or fluorescence microscope, a charge-coupled device camera or imager, a phosphorescent imager, or a combination thereof.

18. The method according to claim 16, characterized in that, The tissue samples were taken from subjects selected from the following: humans, non-human primates, rats, mice, guinea pigs, rabbits, pigs, cattle, sheep, goats, horses, dogs, cats, fish, birds, reptiles, amphibians, insects, plants, fungi, bacteria, or combinations thereof.

19. The method according to claim 16, characterized in that, The analytes include one or more of metabolites, proteins, nucleic acids, carbohydrates, or lipids.

20. The method according to claim 16, characterized in that, The affinity molecule is an antibody, a part of an antibody, an antibody-like molecule, a ligand receptor, a ligand of a receptor, a member of a coupling pair, an aptamer, or an antigen.

21. The method according to claim 16, characterized in that, The affinity molecule is conjugated to a fluorescent molecule, conjugated to an enzyme, bound to another affinity molecule conjugated to a fluorescent molecule, or bound to another affinity molecule conjugated to an enzyme.

22. The method according to claim 16, characterized in that, The affinity molecule is conjugated with the enzyme.

23. The method according to claim 22, characterized in that, The enzyme in question is horseradish peroxidase.

24. The method according to claim 23, characterized in that, The detected signal originated from the tyrosine conjugate.

25. The method according to claim 24, characterized in that, The tyrosamide conjugate comprises one or more of Alexa Fluor 350, 488, 546, 588, 594, 647 or 750 tyrosamide reagents or Biotin-XX tyrosamide reagents.

26. A kit comprising: One or more sample compartmentation modules according to claim 1; One or more affinity molecules; One or more signal generating reagents; Optionally, one or more reagents, including deparaffinizing reagents, enzyme activating reagents, antigen retrieval reagents, sample dilution reagents, reagent dilution buffers, blocking reagents, or endogenous enzyme activity blocking reagents; and Optionally, one or more containers, packaging, instructions for use, MSDS, reference or control tissue samples, or reference or control targets.

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