Method, solution and kit for preparing a tissue sample for 3D imaging

Abstract

The present invention relates to a solution for the preparation of tissue samples for three-dimensional (3D) imaging. The present invention also relates to methods and uses involving the solution.

Description

Technical Field

[0001] The present invention relates to a solution for the preparation of tissue samples for the purpose of three-dimensional (3D) imaging. The present invention also relates to methods and uses in which the solution is involved.

Background Art

[0002] Histology is the field of study of the anatomy of plant and animal cells and tissues using microscopy. It is generally studied using specimens that have been sectioned, stained, and mounted on microscope slides, either with an optical or an electron microscope. The ability to visualize or specifically identify microscopic structures is often enhanced through the use of staining. Histology is an essential tool in biology and medicine.

[0003] Histopathology is the field of study of diseased tissues and is an important tool in anatomical pathology because accurate diagnosis of cancer and other diseases usually requires histopathological examination of samples. Pathologists can perform histopathological examinations and provide diagnostic information based on their observations. Pathologists examine stained tissue specimens by bright-field microscopy to estimate various histological and pathological findings, such as tissue composition, tissue structure and morphology, cell morphology, cell malignancy, degree of inflammation and fibrosis, depth of tumor invasion, presence of tumor components at the resection margin, and lymph node metastasis status.

[0004] Conventional methods of histology and histopathology involve staining and imaging using microscopy. However, while conventional methods provide information regarding morphological changes in cells and tissues, they also have fundamental limitations. For example, conventional methods can only provide planar two-dimensional (2D) images, limiting their ability to observe three-dimensional (3D) structures composed of various cells in diverse anatomical structures. Furthermore, in histopathological diagnosis of particularly large pathological specimens, such as surgically resected specimens, only representative lesions identified by macroscopic observation are generally evaluated. Thus, there remains a concern that additional significant lesions may be present in areas that have not been evaluated. Traditional 2D histological examination of tissues limits appropriate insight into 3D tissue structure. For example, 3D imaging is essential for exploring the connectivity of local cell networks.

[0005] However, it has been found that the preparation of tissue samples for 3D imaging is a challenge. Prior art for preparing tissue samples for 3D imaging has focused on increasing tissue transparency in order to reduce the light scattering properties of multi-cell layers, which are both important for light microscopy, and increase light transmittance. Prior art was developed in neural tissue. Their application increases transparency in other tissues as well, but it is not possible to stain them, particularly by immunolabeling.

[0006] Furthermore, prior art used in the art for the preparation of tissue samples for 3D imaging utilizes various enhanced fixations of the sample, such as hydrogel embedding, because it was considered in the art that such enhanced fixation is necessary to achieve a tissue sample suitable for 3D imaging.

[0007] However, the use of such enhanced fixation is disadvantageous as it degrades the quality of the tissue sample and reduces the number and variety of different antibodies that can be used, for example, in immunolabeling of the sample. It is advantageous to be able to image multiple antibodies of different specificities in a single tissue sample, as the amount of sample available (e.g., in a clinical or diagnostic setting, such as from a biopsy) is often limited.

[0008] Due to the limitations in the number and variety of antibodies that can be used with the resulting tissue samples, this is not possible with current methods in the art for preparing tissue samples for 3D imaging. In this regard, the resulting tissue samples are not compatible with labeling by a wide range of antibody types and specificities. While not wishing to be bound by theory, this may be due to masking of antigens during the enhanced fixation step. SUMMARY OF THE INVENTION PROBLEM TO BE SOLVED BY THE INVENTION

[0009] Accordingly, there is a need in the art for an alternative method of preparing tissue samples for 3D imaging that facilitates the use of a wider range of antibodies, for example, for immunolabeling of samples. MEANS FOR SOLVING THE PROBLEM

[0010] The inventors have surprisingly found that the methods described herein can be used to prepare tissue samples for 3D imaging by immunolabeling, particularly using a wide range of antibodies. The inventors have surprisingly found that a method less complex than those previously used in the art can be used to prepare tissue samples. This example demonstrates that additional fixation with, for example, hydrogel or glutaraldehyde (GA) is not required to achieve a tissue sample suitable for 3D imaging by immunolabeling.

[0011] Thus, the present invention provides a highly advantageous method for preparing tissue samples and a solution that can be used in said method.

[0012] Furthermore, previous techniques for tissue sample preparation used solutions with a pH of 9 or higher because such a pH was thought to be necessary to make the tissue sample transparent for 3D imaging. However, the inventors have surprisingly found that a solution containing a surfactant and a buffer with a pH of less than 9 can be successfully used in the preparation of tissue samples for 3D imaging. The inventors have also found that the solution according to the present invention can be used at a higher temperature than previous techniques.

[0013] The present invention provides a solution containing a buffer with a pH of less than 9 and a surfactant for preparing a tissue sample for 3D imaging.

[0014] As demonstrated in this example, the solution according to the present invention can be used to prepare various tissue samples using various antibodies. Thus, advantageously, the present invention has general applicability in the preparation of tissue samples for 3D imaging. This example also demonstrates that the present invention can facilitate imaging with a resolution equivalent to 2D histochemistry, and that tissue integrity and organ structure are maintained.

[0015] The present invention is also time- and cost-effective and does not require special equipment or sample procurement (such as hydrogel embedding, GA fixation, etc.). This represents a further advantage over previously used techniques in the art. As noted above, while not wishing to be bound by theory, prior art techniques have drawbacks associated with excessive fixation, complex epitope retrieval, and cross-linking that need to be overcome to prepare tissue samples suitable for imaging by achieving tissue integrity, preserving organ structure, and generating reliable signals when immunolabeled.

[0016] Thus, the present invention can enable the use of simpler fixation steps, such as reducing the time the tissue sample is exposed to the fixative, or using conventional fixatives such as neutral buffered formalin (NBF).

[0017] Advantageously, the present invention facilitates the supply of a wide range of tissue samples for 3D imaging using a wide range of antibodies. Traditional histological techniques for 2D imaging may not be effective over a wide range of tissues, and traditional histological techniques may not function with a wide range of antibodies, including antibodies with broad and narrow specificities.

[0018] Another advantage is that the present invention enables imaging of intact tissue samples without the need for serial sections of the sample. Advantageously, the present invention also enables 3D imaging of tissue samples that are partially or serially sectioned to a degree that matches or improves upon traditional histological techniques.

[0019] The ability to provide immunolabeled intact tissue samples for 3D imaging can be particularly advantageous for pathological or diagnostic purposes. For example, the present invention may enable 3D tumor imaging and determination of tumor margins within tissue samples. This is advantageous compared to conventional histological analysis that requires time-consuming and costly serial sectioning of tissue samples.

[0020] In conventional histology for pathological or diagnostic purposes, it is common to sample a portion of the whole tissue, and it is known that inadequate sampling may miss essential markers or the entire tumor, which can lead to inaccurate conclusions regarding the presence or absence of a disease state. Thus, the present invention can advantageously reduce the likelihood that a disease state will not be detected and advantageously provides an improved method for determining the presence or absence of a disease state in a tissue sample, i.e., in a diagnostic setting.

[0021] One advantage is that the method or use of the present invention provides an improved tissue sample for 3D imaging, which enables a wider range of immunolabeling from the tissue sample for 3D imaging.

[0022] As a further advantage, the present invention can also be used for archival tissue samples, such as those in paraffin blocks.

[0023] Tissue samples that are archived (stored) in paraffin blocks are known to be brought into contact with paraffin and embedded therein for storage (paraffin embedding) using standard methods. It is also well known that to retrieve the tissue sample from storage, the stored tissue sample is then deparaffinized and subsequently processed for imaging. Conventional immunolabeling methods are not suitable for use with paraffin-embedded samples, but the invention described herein is surprisingly suitable for use with such samples.

[0024] The solution of the invention described herein can be used in the preparation of tissue samples. As used herein, the term "preparation" as used herein encompasses any manner of generating a tissue sample prior to being subjected to 3D imaging.

[0025] In one aspect, the tissue sample can be derived from a mouse, rat, rabbit, cow, pig or non-human primate. In a preferred aspect, the tissue sample is derived from a human.

[0026] The method of the invention has many uses, for example in medicine and research. The method of the invention can be used to diagnose, determine the presence or absence of, or monitor a disease state.

[0027] The method of the invention can be used to study healthy or diseased tissue, or to investigate the efficacy of a candidate drug in disease modification. Solutions, kits and their use that are useful in the practice of the method of the invention are also provided.

[0028] The solution can also be used for the preparation of three-dimensional cell culture models for staining.

[0029] In one embodiment, the invention provides a method of preparing a tissue sample for 3D imaging, the method comprising treating the tissue sample with a solution comprising a buffer with a pH of less than 9 and a surfactant.

[0030] In another embodiment, the present invention is a method for preparing a tissue sample for 3D imaging, comprising: a) preparing a tissue sample; b) fixing the tissue sample; c) optionally, archiving the tissue sample; d) treating the tissue sample with a solution containing a buffer with a pH less than 9 and a surfactant; e) optionally, immunolabeling the tissue sample. A method including the above steps is provided.

[0031] Preferably, by the method of the present invention, the tissue sample may not be embedded in a hydrogel and / or may not be fixed with glutaraldehyde.

[0032] Preferably, by the method of the present invention, 3D imaging may be based on immunostaining.

[0033] By the method of the present invention, a) the surfactant is SDS or zwitterionic, preferably a Zwittergent® surfactant, and the zwittergent surfactant may be selected from any one of n-octyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid, n-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid, n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid, n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid, and n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid; and / or b) the pH of the buffer may be less than 8.5, less than 8, less than 7.5, or 7; and / or c) the buffer may be a boric acid or citrate buffer, preferably boric acid.

[0034] Preferably, in the method of the present invention, the solution can be used at a temperature between about 40°C and about 60°C, preferably between about 50°C and about 60°C, more preferably at a temperature of about 55°C or about 54°C.

[0035] Preferably, by the method of the present invention, the tissue sample may be: a) a mouse, rat, rabbit, bovine, porcine or non-human primate; or b) derived from a human, and preferably, the tissue sample is a surgically excised specimen.

[0036] Preferably, by the method of the present invention, the tissue sample may be fixed using neutral buffered formalin, preferably 10% neutral buffered formalin.

[0037] Preferably, the method of the present invention further comprises the step of determining the presence or absence of a disease state in the tissue sample.

[0038] Preferably, by the method of the present invention, the tissue sample may be paraffin-embedded.

[0039] Preferably, by the method of the present invention, the tissue sample may be an intact tissue sample.

[0040] In one embodiment, the present invention provides a solution comprising a buffer with a pH of less than 9 and a surfactant for preparing a tissue sample for three-dimensional (3D) imaging.

[0041] Preferably, the 3D imaging can be based on immunostaining.

[0042] Preferably, a) the surfactant may be SDS or zwitterionic, preferably a Zwittergent® surfactant, and the zwittergent surfactant is selected from any one of n-octyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid, n-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid, n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid, n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid, and n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid; and / or b) the pH of the buffer may be less than 8.5, less than 8, less than 7.5, or 7; and / or c) the buffer may be a boric acid or citrate buffer, preferably boric acid.

[0043] Preferably, the solution can be used at a temperature between about 40°C and about 60°C, preferably between about 50°C and about 60°C, more preferably at a temperature of about 55°C or about 54°C.

[0044] Preferably, with the solution, the tissue sample may be: a) from a mouse, rat, rabbit, cow, pig, or non-human primate; or b) derived from a human, and preferably the tissue sample is a surgically excised specimen.

[0045] In one embodiment, the present invention provides a method for preparing a solution according to the present invention, which includes combining the buffer with a pH less than 8 with the surfactant.

[0046] In one embodiment, the present invention provides the use of a solution according to the present invention for preparing a tissue sample for 3D imaging.

[0047] Preferably, the use is for determining the presence or absence of a disease state.

[0048] In one embodiment, the present invention provides a solution comprising a buffer with a pH less than 9 and a surfactant for preparing a tissue sample for three-dimensional (3D) imaging, and the surfactant is Zwittergent®.

[0049] Preferably, the pH of the buffer is less than 9. Preferably, the pH of the buffer is less than 8.5. Preferably, the pH of the buffer is less than 8.

[0050] Preferably, the buffer is not PBS. Preferably, the buffer is boric acid or citric acid.

[0051] In one embodiment, the present invention provides a solution comprising a buffer with a pH less than 8.5, preferably less than 8, and a surfactant for preparing a tissue sample for three-dimensional (3D) imaging, and the buffer is boric acid.

[0052] Preferably, the surfactant is zwitterionic.

[0053] In one embodiment, the present invention provides a solution comprising a buffer with a pH of 7 and a surfactant for preparing a tissue sample for three-dimensional (3D) imaging.

[0054] Preferably, the surfactant is SDS.

[0055] Preferably, the surfactant is zwitterionic or Zwittergent®.

[0056] In one embodiment, the present invention provides a solution comprising a buffer with a pH less than 8.5 and a surfactant for preparing a tissue sample for three-dimensional (3D) imaging, the surfactant is SDS, and the buffer is a boric acid solution. The present invention also relates to the following. [Item 1] A method for preparing a tissue sample for 3D imaging, comprising treating the tissue sample with a solution containing a buffer with a pH of less than 9 and a surfactant. [Item 2] A method for preparing a tissue sample for 3D imaging, comprising: a) preparing a tissue sample; b) optionally, fixing the tissue sample; c) optionally, archiving the tissue sample; d) treating the tissue sample with a solution containing a buffer with a pH of less than 9 and a surfactant; and e) immunolabeling the tissue sample The method comprising. [Item 3] The method according to item 1 or 2, wherein the tissue sample is not embedded in a hydrogel and / or is not fixed with glutaraldehyde. [Item 4] The method according to any one of items 1 to 3, wherein the 3D imaging is based on immunostaining. [Item 5] a) The surfactant is SDS or zwitterionic, preferably a Zwittergent® surfactant, and the Zwittergent® surfactant is selected from any one of n-octyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid, n-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid, n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid, n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid, and n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid; and / or b) The pH of the buffer is less than 8.5, less than 8, less than 7.5 or 7; and / or c) The buffer is a boric acid or citrate buffer, preferably boric acid, The method according to any one of items 1 to 4. [Item 6] The method according to any one of items 1 to 5, wherein the solution is used at a temperature between about 40°C and about 60°C, preferably between about 50°C and about 60°C, more preferably about 55°C or about 54°C. [Item 7] The tissue sample is a) derived from a mouse, rat, rabbit, cow, pig or non-human primate; or b) human The method according to any one of items 1 to 6, preferably the tissue sample is a surgically resected sample, a sample of 3D cell culture material (organoid) or a sample of tissue made by biotechnology. [Item 8] The method according to any one of items 1 to 7, wherein the tissue sample is fixed using neutral buffered formalin, preferably 10% neutral buffered formalin. [Item 9] The method according to any one of items 1 to 8, further comprising determining the presence or absence of a disease state in the tissue sample. [Item 10] The method according to any one of items 1 to 9, wherein the tissue sample is paraffin-embedded. [Item 11] The method according to any one of items 1 to 10, wherein the tissue sample is an intact tissue sample. [Item 12] A solution for preparing a tissue sample for three-dimensional (3D) imaging, comprising a buffer solution with a pH of less than 9 and a surfactant. [Item 13] The solution according to item 12, wherein the 3D imaging is based on immunostaining. [Item 14] a) The surfactant is SDS or zwitterionic, preferably a Zwittergent® surfactant, and the Zwittergent® surfactant is selected from any one of n-octyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid, n-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid, n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid, n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid, and n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid; and / or b) The pH of the buffer solution is less than 8.5, less than 8, less than 7.5 or 7; and / or c) The buffer solution is boric acid or citrate buffer, preferably boric acid. The solution according to item 12 or item 13. [Item 15] The solution according to any one of items 12 to 14, wherein the solution is used at a temperature between about 40°C and about 60°C, preferably between about 50°C and about 60°C, more preferably at a temperature of about 55°C or about 54°C. [Item 16] The tissue sample is a) derived from a mouse, rat, rabbit, cow, pig or non-human primate; or b) human and preferably the tissue sample is a surgically resected specimen, a sample of 3D cell culture material (organoid) or a sample of tissue produced by biotechnology. The solution according to any one of items 12 to 15. [Item 17] A method for preparing the solution according to any one of items 12 to 16, comprising combining the buffer solution with a pH of less than 8 with the surfactant. [Item 18] Use of the solution according to any one of items 12 to 16 for preparing a tissue sample for 3D imaging. [Item 19] Use of the solution according to item 18 for determining the presence or absence of a disease state. [Item 20] Kit for preparing a tissue sample for 3D imaging, comprising the solution according to any one of items 12 to 16.

Brief Description of the Drawings

[0057] [Figure 1] Epitope recovery enables whole - tissue immunolabeling. a) Intact pancreatic lobes were treated for 16 hours at the indicated temperature with the indicated buffer containing 4% SDS. Staining for Krt19 (pancreatic ducts) is shown. The blue line indicates the lowest temperature above which staining was observed; the red line indicates the highest temperature above which sample damage was noted. The × marks indicate sample loss. [Figure 2] Tissue clearing is not sufficient for immunolabeling. a) Immunofluorescent staining for amylase (acinar cells), PCSK1 (islets of Langerhans), and SMA (stroma and vasculature) in paraffin - embedded, sectioned pancreas (4 μm) after heat - mediated antigen retrieval. Scale bar 100 μm. b) Staining with the same antibodies as in (a) in untreated control (PBS) pancreas and pancreas cleared with iDISCO, Clarity, and CUBIC buffers. 3D images of representative regions are shown. Scale bar 100 μm. c) 3D reconstruction of pancreas stained with the indicated antibodies 16 hours after FLASH treatment. [Figure 3]FLASH is a figure showing the ability to perform immunostaining of the lung. a-d) Staining for CC10 (Clara cells) and SMA (myoepithelial cells and vasculature). a) 3D image of an intact lung lobe showing the bronchiolar tree. Scale bar 1 mm. b) Magnification of the area (white box) indicated in (a) showing the complex arrangement of myoepithelial cells extending around the bronchioles. Scale bar 500 μm. c) Magnified image of the area (white box) indicated in (b). Scale bar 150 μm. c') Optical section demonstrating the intact compartmentalization of the epithelial and myoepithelial tissue layers. Scale bar 50 μm. d) Comparative staining of paraffin-embedded lung tissue sections (4 μm). Scale bar 50 μm. e) Staining for CC10, SMA, and Sp-C (type II alveolar cells). Scale bar 100 μm. [Figure 4] FLASH is a figure showing the ability to perform immunostaining of the liver. a) 3D image of a liver segment stained for GS (pericentral hepatocytes). Scale bar 300 μm. b) 3D reconstruction and optical section (b') of bile ducts stained for Krt19 (duct cells) and Aqp1 (microcirculation system). Scale bar 200 μm. c) Staining for Krt19 and Prox1 (lymphatic endothelium, nuclei). Scale bar 100 μm. d) Bile ducts stained with DBA (duct cells) and CD44 (bile duct cells). Scale bar 50 μm. d') Optical section of the duct shown in (d) demonstrating the maintained localization of CD44 to the lateral cell membrane. Scale bar 20 μm. [Figure 5] FLASH is a figure showing the ability to perform immunostaining of the lacrimal gland. a) 3D reconstruction of the lacrimal gland stained for Krt19 (duct cells), SMA (stroma), and Aqp1 (microcirculation system). Scale bar 200 μm. b) Staining for Krt19 and S100 (nerves). Scale bar 200 μm. c) Staining for S100 and Krt14 (myofibroblasts). Scale bar 100 μm. d) Staining for Krt19 and vimentin (fibroblasts). Scale bar 200 μm. d') Optical section through the image in (d) demonstrating the compartmentalization of the mesenchymal and epithelial tissue layers. Scale bar 100 μm. [Figure 6]FLASH is a figure showing the ability to enable immunostaining of the kidney. a) 3D reconstruction of the kidney stained for DBA (collecting ducts and groups of tubules) and PNA (distal tubules). Scale bar 1 mm. b) Staining for DBA, PNA and WT1 (glomerulus, nucleus). Scale bar 100 μm. b') Optical section through the area (white box) indicated in (b). Scale bar 50 μm. c) Staining for DBA, PNA and WT1 on paraffin-embedded kidney sections (4 μm). Scale bar 50 μm. [Figure 7] FLASH is a figure showing the preserved intracellular protein localization after FLASH. a) 3D image of the FLASH-treated pancreas showing the arrangement of acinar cells (Amy, amylase) in groups (acini) between sheets of CollIV-positive extracellular matrix. Scale bar 50 μm. a') Optical section through the area of (a) showing Mist1 (acinar cell, nucleus), Amy and CollIV localization in one acinus. Scale bar 20 μm. b) Fluorescence intensity along the indicated line of (a) showing different intracellular localizations of the epitope. c) One-channel image of (a'). d) Staining for Amy, CollIV and Mist1 on paraffin-embedded pancreatic tissue sections (4 μm). Scale bar 20 μm. e) Intensity profile of the fluorophore in (d). [Figure 8] FLASH is a figure showing the intact tissue morphology after FLASH. a) Hematoxylin & eosin (H&E) staining on paraffin-embedded PBS control pancreas, liver and lung (4 μm tissue sections). b) H&E staining on FLASH-treated pancreas, liver and lung (4 μm tissue sections) demonstrating the suitability of the use of FLASH-treated samples for preserved tissue morphology and subsequent 2D staining and histological analysis. All scale bars 100 μm. [Figure 9-1]Figure showing FLASH imaging of the intact pancreas and visualization of the ductal tree. (a–c) 3D rendering of tdTomato-stained intact pancreas from tamoxifen-treated R26-CAG-tdTomato;Hnf1βCreERT2 mice. (a) 3D image of the whole pancreas attached to the duodenum and spleen. Scale bar 5 mm. (b) 3D magnification of the area in (a). Scale bar 500 μm. (c) 3D image of duct segments. (1) Main duct; (2) interlobular duct; (3) intralobular duct; (4) intercalated duct. Scale bar 100 μm. (d) Segment heterogeneity of duct diameters. Each point represents one duct, 180 ducts, 3 mice. (e) Pancreatic tissue section stained for Cdh1 and DNA to highlight different duct cell shapes. Scale bar 10 μm. (f) Segment heterogeneity of duct cell width (black), height (red), and length (blue); average of 5 cells per duct, 115 ducts, 6 mice. Fitted lines were obtained using non-linear regression. (g) Illustration of segment heterogeneity of the ductal tree. L - cell length, W - cell width, H - cell height. [Figure 9-2] Continuation of FIG. 9-1. [Figure 9-3] Continuation of FIG. 9-2. [Figure 10-1]A figure showing the integrity of the preserved organs after FLASH. (a) FLASH staining of the pancreas of an insulin (Ins)-GFP reporter mouse for amylase (Amy), Krt19, and Ins-GFP. Left, 3D reconstruction demonstrating the complex tissues of the islets and pancreatic ducts. Scale bar 100 μm. Right, optical section through the indicated region (left) showing the preserved compartmentalization into exocrine and endocrine glands as seen by mutually exclusive staining for Amy, Krt19, and Ins-GFP. Scale bar 50 μm. (b) 3D image of a high-diameter duct (32 μm diameter) stained for Krt19 and DNA demonstrating preserved epithelial integrity. Scale bar 50 μm. (b') Left, optical section through the indicated region of (b) demonstrating a continuous monolayer of duct cells and a preserved duct lumen. Scale bar 30 μm. Right, staining for Krt19 and DNA in a 4 μm tissue section of paraffin-embedded pancreas. Scale bar 30 μm. (c) FLASH staining for tdTomato (tdTOM) and Krt19 in the pancreas of R26-CAG-tdTomato;Hnf1βCreERT2 mice without (left) and with (right) an intraperitoneal injection of 100 μg tamoxifen per gram body weight. Scale bar 500 μm. (d) Dendritic branching of the duct tree. Each point represents one duct and the lines indicate branches. For three Krt19-stained pancreases, z-stacks of at least 30 random high-magnification images were taken. To classify the mode of dendritic branching, the largest duct for each image was identified and the diameters were measured for the four subsequent branching ducts. For ducts with multiple branches, the sequence was continued with the largest duct from which they branched. To indicate the end of the duct tree, a duct diameter of 0 μm was assigned to the terminal duct cells. (e) Direction of cell division measured as the angle determined with respect to the line connecting the nucleus and the direction of the duct in 2-cell clones from tamoxifen-treated R26-LSL-Confetti;Hnf1β-CreERt2 mice. (263 clones, 5 mice). (f) Direction of cell division and aspect ratio in ducts of various diameters. Solid lines represent exponential fits. [Figure 10-2] Continuation of Figure 10-1. [Figure 11]A figure showing the heterogeneity of neoplasm induction in the pancreatic duct. (a) Deformation of the outer proliferative and inner proliferative pancreatic ducts. (b - g) FLASH comparison by 2D histology. (b, e) 3D images (left) and optical sections (right) of the outer proliferative (b) and inner proliferative (e) deformations of KrasG12D;Fbw7 F / F;R26 - EYFP;Ck19 - CreERt (KFCk19) 10 days after recombination. Scale bar 50 μm. (c - d, f - g) Hematoxylin - eosin (H&E) staining of the outer proliferative (c - d) and inner proliferative (f - g) lesions 10 days (c, f) and 21 days (d, g) after recombination. Scale bar 100 μm. [Figure 12-1]Figure showing exophytic and endophytic neoplasms in the KrasG12D;Fbw7 F / F model. (a) KFCk19 mouse model of tumor induction in ductal epithelium induced by Fbw7 exon deletion and KrasG12D activation. (b) The recombination efficiency of low-dose tamoxifen injection and the number of transformed clones per recombined cell were quantified 1 week after tamoxifen injection in KFCk19 mice. The total number of duct cells was estimated by quantifying EYFP-traced Krt19+ cells per duct, dividing the duct length by the average cell length measured for this duct, and multiplying this by the average number of cells circumscribing this duct. Transformed clones were recognized as groups of more than 3 EYFP-traced cells sharing an interface. One point represents one duct (112 ducts, 3 mice). (c-d) KFH mouse model for targeting instead of pancreatic ducts. (d) Left, 3D renderings of exophytic neoplasms (top) and endophytic neoplasms (bottom) in KFH mice. Staining for Krt19 and tdTomato. Scale bar 100 μm. Right, H&E staining for exophytic (top) and endophytic (bottom) lesions in KFH. Scale bar 100 μm. (e) H&E and AB / PAS staining for exophytic and endophytic lesions in KFCk19 mice demonstrating the common non-mucinous nature of duct-derived neoplasms. Scale bar 100 μm. (f) Experimental strategy for visualizing the relationship of exophytic neoplasms to the duct tree. A cannula was inserted into the hepatic extrahepatic bile duct at the dilated part and 50 μl of FITC-labeled dextran was perfused into the pancreatic duct tree. (g) DexFITC uptake by exophytic KFCk19 lesions demonstrating the relationship of the lesion to the duct system. Left, 3D image, and right, optical section. Scale bar 50 μm. [Figure 12-2] Continuation of Figure 12-1. [Figure 12-3] Continuation of Figure 12-2. [Figure 13-1]Figure showing the morphological progression of neoplasms derived from acinar cells. (a) Schematic diagram illustrating the genetic strategy for acinar cell transformation by KrasG12D activation through simultaneous p53 F / F or Fbw7 F / F deletion using Ela1-CreERt or Ptf1a-ERt2 drivers. (b–c) KrasG12D;Fbw7 F / F;Ela1-CreERt mice (KFEla1). (b) 3D image of acinar-to-ductal dysplasia identified by local upregulation of Krt19 expression in acinar cells. td-Tomato traced acini connected to terminal ducts are shown. The arrow tip demarcates Krt19 expression by the central acinus and forms a small ring of acinar-derived td-Tomato traced Krt19+ cells. Scale bar 50 μm. (c) 3D projection of a spherical KFEla1 lesion in contact with small-diameter ducts (dotted line). Scale bar 20 μm. (c') Optical section of the lesion shown in (c) demonstrating td-Tomato tracing (top) and spherical morphology (bottom). Scale bar 20 μm. (d–g) KrasG12D;p53 F / F;Ela1-CreERt mice (KPEla1). (d) 3D image of a spherical td-Tomato traced KPEla1 lesion connected to a terminal duct (arrow tip). Scale bar 50 μm. (e) 3D projection of a large KPEla1 lesion showing the central grape-like morphology of the back-to-back spherical structures and maintained connection to some small-diameter ducts (arrow tips) at the edge of the lesion. Scale bar 200 μm. (e') Higher magnification of the indicated region in (e) demonstrating seamless connection of acinar-derived Krt19+ cells and wild-type duct epithelium (dotted line). Scale bar 30 μm. (f) Retrograde perfusion of the ductal tree with dextran-FITC as in Extended Data Fig. 2f demonstrating direct connection of an acinar-derived lesion to the ductal system. 3D image of the KPEla1 lesion. Scale bar 50 μm. (g) H&E staining of a KPEla1 lesion demonstrating spherical morphology. Scale bar 100 μm. (h) KrasG12D;p53 F / F;Ptf1a-CreERt2 (KPPtf1a) mice. H&E staining demonstrating the spherical morphology of the lesion. Scale bar 100 μm. [Figure 13-2] Continuation of Fig. 13-1. [Figure 14-1]Figure showing KrasG12D;p53 F / F model and exophytic and endophytic neoplasms in the human pancreas. (a-c) Endophytic and exophytic lesions induced by p53 deletion due to KrasG12D activation after Pdx1-Cre-induced whole pancreas recombination (KPC). (b) 3D image of the pancreatic region with endophytic (1) and exophytic (2) deformations. Scale bar 150 μm. (1-2) Higher magnification of the regions indicated in (b). Scale bar 50 μm. (1) The arrow tip defines the invagination typical of endophytic growth. (2) The dotted line marks the morphologically normal small ducts in contact with the spherical exophytic lesions. (c) Hematoxylin-eosin (H&E) staining for exophytic (left) and endophytic (right) lesions in the KPC model. Scale bar 100 μm. (d-e) Exophytic and endophytic lesions induced by p53 deletion due to KrasG12D activation in pancreatic ducts (Ck19-CreERt;KPCk19). (e) 3D projection of the exophytic (left) and endophytic (right) lesion shapes in KPCk19 mice. Scale bar 100 μm. (f) H&E staining of tissue sections from the background pancreas of a patient presenting with pancreatic ductal adenocarcinoma. (Left) Exophytic lesion. (Right) Endophytic lesion. Scale bar 100 μm. [Figure 14-2] Continuation of Figure 14-1. [Figure 15] Figure showing FLASH 3D imaging of human biopsies. (a-b) Exophytic lesions of the human pancreas. (a) H&E staining of a tissue section from the human pancreas. Scale bar 100 μm. (b) FLASH-imaged human pancreas biopsy showing exophytic ductal lesions identified by Krt19 immunolabeling. Left, 3D image. Right, optical section. (c-d) Endophytic lesions. (c) H&E staining of the human pancreas. Scale bar 100 μm. (d) FLASH-imaged human pancreas biopsy showing endophytic ductal lesions identified by Cdh1 immunolabeling (green). Muc5AC immunostaining (red) identifies mucinous cells and SMA staining (white) identifies the surrounding stroma. Left, 3D image. Right, optical section.

Mode for Carrying Out the Invention

[0058] 3D Imaging The present invention enables visualization of molecular labeling structures in large intact tissues in three dimensions.

[0059] 3D imaging can be performed by methods known to those skilled in the art. For example, 3D imaging can be performed by optical microscopy, fluorescence microscopy, such as confocal microscopy, light sheet microscopy, super-resolution microscopy, spectral precision distance microscopy, activation-induced emission depletion, expansion microscopy, or optical projection tomography and any variants thereof.

[0060] In a preferred embodiment of the present invention, as described herein, 3D imaging is based on immunostaining.

[0061] The present invention facilitates or enables immunostaining of tissue samples for 3D imaging. The present invention facilitates antibody staining of tissue samples. The present invention is based on membrane solubilization by antigen activation without disrupting tissue structure. Unlike prior art, as demonstrated herein, the present invention enables robust staining of various tissue types with a high signal-to-noise ratio.

[0062] As used herein, "immunostaining" refers to any use of antibody-based methods for detecting specific proteins in a sample, such as immunolabeling of tissue sections or immunohistochemical staining.

[0063] In one aspect, the present invention facilitates immunostaining of tissue samples prior to 3D imaging. Immunohistochemistry, or IHC, or immunolabeling of tissue sections (or immunocytochemistry or immunofluorescent labeling which is staining of cells) is perhaps the most commonly applied immunostaining technique. Immunolabeling and immunofluorescent labeling use fluorescent dyes, while immunohistochemistry and immunocytochemistry use enzymes, such as peroxidase and alkaline phosphatase. These enzymes can catalyze reactions that give colored products that are easily detectable by optical microscopy. Alternatively, radioactive elements can be used as labels and the immune reaction can be visualized by autoradiography.

[0064] When immunostaining a specific antigen, several methods can be used. For example, in one method, a label (conjugate) can be prepared by directly binding a fluorescent label and a primary antibody, and then the antigen is stained (primary antibody method). Alternatively, a label can be prepared by directly binding a fluorescent label and a secondary antibody, and the antigen bound to the primary antibody is then stained (secondary antibody method). In yet another method, a label can be prepared by directly binding a fluorescent label and biotin, and the antigen bound to the primary antibody and a secondary antibody modified with avidin or streptavidin is then stained (biotin-avidin method or sandwich method).

[0065] Any suitable primary antibody can be used in immunostaining, and the primary antibody varies depending on the object to be immunostained. For example, when immunostaining is performed using HER2 as an antigen, an anti-HER2 antibody is used. Those skilled in the art will know suitable antibodies for staining.

[0066] In one aspect, the antigen can be selected from SST, KCNE3, PP, C-peptide, Ins, CD44, SMA, RFP and its derivatives, GFP and its derivatives, Krt19, Krt5, Krt14, Krt7, Krt76, pan-cytokeratin, WT-1, Epcam, Muc1, Muc5Ac, Muc2, Prox1, Cdh1, Mist1, Lyz, GFAP, TH, PGC, GIF, Endomucin, PGP9.5, PCSK1 / 3, GS, S100, Aqp1, Aqp2, Gluc, H / K-ATPase, Amy, CC10, SFTPC, CollIV, Vim, Ki67, PCNA, myosin, phosphohistone H3, and cleaved caspase-3.

[0067] Furthermore, any secondary antibody can be used, and the secondary antibody varies depending on the primary antibody. Examples thereof include anti-mouse, rabbit, bovine, goat, sheep, dog, and chicken antibodies.

[0068] Any existing method can be used for the binding of a fluorescently labeled antibody or to biotin. For example, amidation by reaction between an amine and a carboxylic acid, sulfidization by reaction between a maleimide and a thiol, imination by reaction between an aldehyde and an amine, or amination by reaction between an epoxy and an amine can be used.

[0069] Immunostaining is not limited to tissue staining and can also be applied to cell staining.

[0070] In one aspect of the present invention, the staining is not limited to antibodies and any substance that interacts with the sample can be used. For example, dyes that interact with biochemical structures, such as DNA intercalating dyes like Dapi, Syto11, propidium iodide, Draq5, can be used, or agents that interact with the biochemical properties or reactive groups of tissue components, such as lectins like dolichos biflorus lectin, wheat germ cell agglutinin, jacalin or ulex europaeus lectin, which detect certain post-translational modifications in proteins. Detection is not limited to fluorescent labels conjugated to antibodies and can be combined with inherently fluorescent dyes or secondary labels by click-it chemistry.

[0071] In one aspect, 3D imaging is not limited to staining and can include detection of signals specific to the sample, such as second harmonic generation and imaging of fluorescent proteins.

[0072] Buffer A buffer (more precisely, a pH buffer or hydrogen ion buffer) is an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa. When a small amount of a strong acid or base is added to it, the pH of the buffer changes only slightly. Buffer solutions are used as a means to keep the pH at a nearly constant value in various applications.

[0073] The buffer according to the present invention may be any suitable buffer having a pH of less than 9 for preparing tissue samples for 3D imaging.

[0074] In one aspect, the buffer may be selected from citric acid, acetic acid, borate, CHES, KH2PO4, Na2HPO4, TAPS ([tris(hydroxymethyl)methylaminopropanesulfonic acid]), Bicine (2-(bis(2-hydroxyethyl)amino)acetic acid), Tris (tris(hydroxymethyl)aminomethane), or (2-amino-2-(hydroxymethyl)propane-1,3-diol), Tricine (3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid), TAPSO (3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TES (2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), cacodylate (dimethylarsinic acid), MES (2-(N-morpholino)ethanesulfonic acid), and combinations thereof. In one aspect, the buffer may be phosphate buffered saline (PBS).

[0075] In a preferred aspect, the buffer is a boric acid or citric acid buffer solution.

[0076] In one aspect, the boric acid solution has a concentration of about 50 - 500 mM, such as about 50, 100, 200, 250, 300, 350, 400, 450, or 500 mM. In one aspect, the concentration is about 200 mM.

[0077] In one aspect, the pH of the buffer is in the range from about pH 3 to less than pH 9. In one aspect, the pH of the buffer is in the range from about pH 4 to less than pH 9. In one aspect, the pH of the buffer is in the range from about pH 5 to less than pH 9. In one aspect, the pH of the buffer is in the range from about pH 6 to less than pH 9. In one aspect, the pH of the buffer is in the range from about pH 7 to less than pH 9. In one aspect, the pH of the buffer is in the range from about pH 8 to less than pH 9.

[0078] In one aspect, the pH of the buffer is about 8.9, 8.8, 8.7, 8.6, 8.5, 8.4, 8.3, 8.2, 8.1, 8.0, 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1 or 3.0.

[0079] In a preferred aspect, the pH of the buffer is about 7.0.

[0080] Temperature In one aspect, the solution according to the present invention is for use at a temperature between about 30°C and about 100°C or should be used. For example, the solution can be used at a temperature between about 40°C and 95°C, about 50°C and about 90°C, about 55°C and 85°C, about 60°C and about 80°C, or about 65°C and 75°C. In a preferred aspect of the present invention, the solution is used at a temperature between about 50°C and 60°C, preferably about 55°C. The solution according to the present invention can be used at a temperature selected from about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 and 95°C.

[0081] Surfactant The surfactant suitable for use in the present invention is the surfactant used in standard methods of tissue sample preparation.

[0082] In one aspect, the surfactant is selected from non-ionic, ionic or zwitterionic surfactants. In one aspect, the surfactant is an ionic surfactant. In one aspect, the surfactant is a zwitterionic surfactant.

[0083] Nonionic surfactants can be selected from BigCHAP (N,N-bis[3-(D-gluconamido)propyl]colamide), Brij® 35 (polyethylene glycol dodecyl ether), C12E8 (octaethylene glycol monododecyl ether), C12E9 (polyoxyethylene(9)dodecyl ether), decyl-β-glucoside, decyl-β-maltoside, deoxy-BigCHAP (N,N-bis[3-(D-gluconamido)propyl]deoxycholamide), digitonin, dodecyl-β-glucoside, dodecyl maltoside, lubrol PX, Nonidet® P-40 (octylphenoxypoly(ethyleneoxy)ethanol, branched), octyl-β-glucoside, octyl-β-maltoside, octyl-β-thiogalactoside, octyl-β-thioglucoside, PLURONIC® F-127 (polyoxypropylene polyoxyethylene block copolymer), Triton® X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol), Tween® 20 (polyethylene glycol sorbitan monolaurate) and Tween® 80 (polyethylene glycol sorbitan monooleate).

[0084] In one aspect of the present invention, the surfactant is an ionic surfactant. The ionic surfactant can be selected from sodium cholate, CTAB (cetyltrimethylammonium bromide), sodium deoxycholate, lithium sulfate, sodium taurocholate and sodium taurodeoxycholate. Preferably, the anionic surfactant is sodium dodecyl sulfate (SDS).

[0085] In an alternative aspect, the surfactant is an amphoteric surfactant. The polar head group of the amphoteric surfactant contains both negatively and positively charged atomic groups, and thus the overall charge is neutral. The intensity of the action of these compounds is thought to be intermediate between ionic and nonionic surfactants and share the characteristics of both types.

[0086] The zwitterionic surfactant can be selected from CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid hydrate) and CHAPSO (3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonic acid), which are commercially available, for example, from Merck.

[0087] In one aspect, the zwitterionic surfactant is a Zwittergent® surfactant that can be selected from Zwittergent® 3-08 (n-octyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid), Zwittergent® 3-10 (n-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid), Zwittergent® 3-12 (n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid), Zwittergent® 3-14 (n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid), and Zwittergent® 3-16 (n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid).

[0088] In a preferred embodiment of the present invention, the surfactant is Zwittergent® 3-10 (n-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid).

[0089] The Zwittergent® surfactant is commercially available, for example, from Calbiochem (Merck KGaA, Darmstadt, Germany).

[0090] One skilled in the art can determine the amount of surfactant contained in the solution according to the present invention.

[0091] In a preferred embodiment, 4% SDS or 8% Zwittergent® 3-10 can be used.

[0092] Additive To further enhance staining, compounds that assist in antigen activation or increase sample penetration can be added to the solution. Compounds that support antigen activation can be, for example, formaldehyde scavengers such as ascorbic acid, urea, Tris, 2-imidazolidinone, or catalysts such as anthranilic acid and phosphanilate, or proteases such as trypsin or pepsin or collagenase. Compounds that support sample penetration can be chaotropic agents such as ammonium thiocyanate, n-butanol, dimethyl sulfoxide, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, urea, magnesium chloride, phenol, 2-propanol, sodium thiocyanate and thiourea, or reducing agents such as 1,4-dithiothreitol, b-mercaptoethanol and tris(2-carboxyethyl)phosphine hydrochloride.

[0093] Tissue sample The invention described herein can be applied to any tissue sample.

[0094] The tissue sample can be selected from epithelial, connective, muscular and nervous tissues.

[0095] In a preferred embodiment, the tissue sample is selected from pancreatic tissue, brain tissue, bone tissue, bone marrow tissue, lung tissue, liver tissue, stomach tissue, breast tissue, head and neck tissue, intestinal tissue, salivary gland tissue, nerve tissue, ovarian tissue, testicular tissue, muscular tissue and skin tissue.

[0096] In one aspect of the invention, the tissue sample can be an embryonic tissue sample.

[0097] In one aspect, the tissue sample can be a diseased tissue sample.

[0098] In one aspect, the tissue sample may be a tumor sample. The tumor may be derived from squamous cell carcinoma or carcinoma, lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric or abdominal cancer, such as gastrointestinal cancer, pancreatic cancer, glioma, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, melanoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, renal or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer or head and neck cancer.

[0099] In one aspect, the tissue sample is not a brain tissue sample.

[0100] In one aspect, the tissue sample is an intact tissue sample.

[0101] In one aspect, the tissue is whole tissue or a part of tissue.

[0102] In one aspect, the tissue sample is not sectioned serially.

[0103] In one aspect, the tissue sample may be fresh, fixed or frozen.

[0104] A fixed tissue sample is a tissue sample that has been treated with any suitable fixative to preserve the tissue from decay. Examples of fixatives include 10% neutral buffered formalin (NBS) solution, 4% paraformaldehyde or glutaraldehyde solution.

[0105] A frozen tissue sample is a tissue sample that has been cryopreserved. Examples of tissue freezing include tissue immersion in isopentane / liquid nitrogen or rapid freezing with dry ice or liquid nitrogen.

[0106] In one aspect, the tissue sample is a sentinel lymph node biopsy.

[0107] Sentinel lymph node biopsy is a surgical procedure used to determine whether cancer has spread beyond the primary tumor into the lymphatic system.

[0108] Method The solution according to the invention described herein can be used in a method for preparing a tissue sample for 3D imaging.

[0109] The method according to the invention may further comprise determining the presence or absence of a disease state in the tissue sample.

[0110] "Disease state" generally means that the method can be used to determine whether a subject is suffering from one or more known diseases. Diseases include, but are not limited to, cancer, autoimmune diseases, inflammatory diseases, metabolic diseases, neurodegenerative diseases, endocrine / reproductive system diseases, cardiovascular / lung diseases, musculoskeletal diseases or gastrointestinal diseases.

[0111] Thus, in certain embodiments, the method of the invention can be used to diagnose the presence of one or more known diseases. This can include obtaining a tissue sample from one or more patients; preferably detecting the presence of one or more diseases using antibody staining; and / or monitoring the progression of one or more diseases in a subject. The method can be used to distinguish a disease from other diseases.

[0112] In embodiments of the invention, the tissue sample is stained with an antibody and 3D imaged. The sample is evaluated for abnormal or unusual immunostaining to determine the presence or absence of a disease state in the tissue sample.

[0113] The method according to the invention is envisioned to be used as a step in determining whether a patient is responding or has responded to a treatment regimen by preparing a tissue sample for 3D imaging using the solution according to the invention.

[0114] Thus, the present invention provides a method for preparing a tissue sample for 3D imaging, the method comprising the step of treating the tissue sample with the solution described herein.

[0115] The present invention also provides the use of the solution described in the invention for preparing a tissue sample for 3D imaging.

[0116] The solution of the present invention can be used to clear, i.e., make transparent, a tissue sample.

[0117] The method according to the present invention can optionally include additional steps.

[0118] The method can include a step of fixing the tissue sample. The method can include a step of washing the tissue sample. The method can include a step of incubating the tissue sample in the solution according to the present invention. The method can include a step of immunolabeling.

[0119] The method can include any one or more of the following steps: 1. Remove the tissue and perform vascular perfusion with PBS if necessary. 2. Fix the tissue. 3. Long-term tissue storage if necessary. 4. Washing step. 5. If applicable, a step of removing debris and adjacent tissue from the sample. Optionally, the sample may be divided into smaller fragments. 6. Incubate the tissue in a solution that enables immunolabeling. 7. Incubation with a primary or secondary antibody.

[0120] In one aspect, the method includes incubating the tissue sample with the solution according to the present invention at a temperature between about 30°C and about 100°C. For example, the incubation can be at a temperature between about 40°C and 95°C, between about 50°C and about 90°C, between about 55°C and 85°C, between about 60°C and about 80°C, or between about 65°C and 75°C. In a preferred aspect of the present invention, the incubation can be at a temperature between about 50°C and 60°C, preferably about 55°C. The incubation can be at a temperature selected from about 30, 35, 40, 45, 50, 54, 55, 60, 65, 70, 75, 80, 85, 90, and 95°C. In one aspect of the invention described herein, the incubation can be at a temperature of about 54 or 55°C.

[0121] The incubation period may be between about 2 and about 48 hours, such as about 6 - 44, 10 - 40, 14 - 36, 18 - 32, or 22 - 28 hours. In one embodiment, the incubation period is about 16 hours. The incubation period may be several days, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In one embodiment, the incubation period is about 24 hours. In one embodiment, the incubation period may be several weeks, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks.

[0122] In one embodiment of the present invention, the solution can be used to prepare a tissue sample for 3D imaging before processing the sample for 2D staining. For example, the tissue sample can undergo 3D imaging before being processed for 2D imaging. In one embodiment, the tissue sample can be prepared using the solution according to the present invention before embedding the sample in paraffin for 2D staining and / or imaging.

[0123] In one embodiment of the present invention, the solution can be used in a method for preparing a tissue sample for 3D imaging for the analysis of human patient materials in a clinic.

[0124] In another embodiment of the present invention, the solution can be used to prepare a tissue sample for 3D imaging for veterinary medicine.

[0125] In another embodiment of the present invention, the solution can be used to prepare a tissue sample for 3D imaging for the analysis of engineered or printed biological tissues.

[0126] Kit The present invention also encompasses a kit comprising the solution according to the present invention for the preparation of a tissue sample for 3D imaging.

[0127] The kit can also include components for facilitating immunolabeling of the tissue sample, such as one or more antibodies.

[0128] The present invention will now be further described by way of examples, which serve to assist those skilled in the art in practicing the present invention and in no way limit the scope of the present invention.

[0129] [Example 1] Materials and Methods FLASH. FLASH was developed for the rapid detection of multiple antigens in intact adult organs by light non-destructive epitope retrieval (rapid optical microscopy analysis of whole organs stained with FLASH-antibodies).

[0130] Mice were euthanized by cervical dislocation. Cardiac perfusion was performed with 20 ml of PBS. Organs were harvested and fixed in 10% NBF at 4°C overnight. Specimens were washed twice with PBT (0.4% Triton X-100 (Sigma-Aldrich) in PBS) for 1 hour. Samples were incubated in a solution of the indicated composition containing 4% SDS for 16 hours for buffer comparison in Figure 1. For FLASH staining of various organs shown in Figures 2-5, specimens were incubated at 54°C for 16 hours in 200 mM boric acid (Sigma-Aldrich) and 4% SDS (Sigma-Aldrich) pH 7.0. For the results shown in Figures 6 and 7, samples were incubated at 54°C for 24 hours in 200 mM boric acid (Sigma-Aldrich) and 8% Zwittergent® 3-10 (Merck) pH 7.0. For Figure 8, samples were incubated at 54°C for 24 hours in PBS (control) or 200 mM boric acid (Sigma-Aldrich) and 8% Zwittergent® 3-10 (Merck) pH 7.0.

[0131] The samples were washed with PBT for more than 3 hours and the volume was exchanged at least three times. For immunolabeling, the samples were incubated in FLASH blocking buffer (1% bovine serum albumin (Sigma-Aldrich), 5% DMSO (Sigma-Aldrich), 10% fetal bovine serum (Gibco), 0.02% sodium azide (Sigma-Aldrich), 0.2% Triton X-100 in PBS) for 1 hour and then incubated with the antisera (all 1:100) on a nutator at room temperature for at least 16 hours. The samples were washed three times with PBS with volume exchange and incubated with the secondary antibodies (all 1:100) at room temperature for at least 2 days.

[0132] The samples were washed with PBS by three volume exchanges, gradually dehydrated in 30%, 50%, 75%, 2×100% MetOH (Sigma-Aldrich) for 1 hour each, and immersed in methyl salicylate diluted with MetOH in a glass dish: 25%, 50%, 75%, 2×100% methyl salicylate (Sigma-Aldrich) for 30 minutes each under light-shielded conditions.

[0133] The fluorescent protein was stained by immunofluorescence. The following antibodies were used: Aqp1 (rabbit, Atlas), Amylase (goat, SCBT), CC10 (goat, SCBT), CD44 (rat, Chemicon), CollIV (rabbit, USBiological), GFP (goat, Abcam), GFP (mouse, Roche), GS (rabbit, Abcam), Krt14 (mouse, Abcam), Krt19 TROMA III (rat, DSHB), Pcsk1 (rabbit, Millipore), Mist1 (mouse, SCBT), Prox1 (rabbit, Abcam), S100 (rabbit, Dako), Sftpc (rabbit, Millipore), SMA (mouse, Sigma-Aldrich), Tomato (rabbit, Rockland), Vim (rabbit, NEB), WT-1 (rabbit, SCBT). All secondary antibodies were Alexa-dye conjugates (ThermoFisher). The nuclei were stained with DRAQ5 (Biostatus). The following lectins were used: DBA-FITC (VectorLabs), DBA-rhodamine (VectorLabs), PNA-FITC (VectorLabs).

[0134] Results To enable immunolabeling of intact tissues, a combination of tissue permeabilization and partial reversal of protein cross-linking was developed to restore antigenicity without compromising tissue integrity. A range of different buffer systems were tested in combination with membrane solubilization and mild heat, followed by antibody incubation in a solvent-rich blocking reagent. The delicate, highly compartmentalized structure of the pancreas is inherently difficult to process for histology because it is vulnerable to damage by pancreatic juice rich in digestive enzymes. After incubation in buffer in a single overnight antigen activation step, whole pancreatic lobules were processed and pancreatic ducts were labeled. Notably, all the buffer systems tested gave robust staining of the branched ducts across the pancreatic lobules. Mild heat was required to achieve staining, but higher temperatures destabilized tissue integrity and caused sample loss (Figure 1). The whole pancreas was then processed with FLASH and subsequently stained with amylase, Pcsk1, and Sma antibodies that had previously failed in conventional cleared pancreas. All staining produced robust signals detectable in three dimensions using standard confocal microscopy, consistent with the performance of the antibodies in traditional 2D staining (Figure 2).

[0135] FLASH was successfully applied to the lung, liver, and lacrimal gland without further technical adaptation, resulting in three-dimensional labeling distributions that were highly consistent with those presented in 2D tissue staining (Figures 3-5). The range of antibodies supported enabled visualization of the structures of all tissue components, sometimes simultaneously, including ducts, vasculature, stroma, nerve distribution, and lymphatic system, as well as tissue-specific cell types such as hepatocytes, pancreatic acini, and alveolar cells (Figures 3-5).

[0136] To further expand the range of suitable staining reagents to antibodies that may be sensitive to residual SDS in FLASH-treated samples, alternative membrane-permeable surfactants were tested. The zwitterionic surfactant Zwittergent-3-10 (FLASH reagent 2) produced robust immunostaining in all tissues analyzed and improved the performance of difficult antibodies such as those against the transcription factors WT1 and Mist1. Importantly, tissue and cell integrity were preserved, and the intracellular staining distribution correlated with traditional 2D immunofluorescence as before (Figs. 6-7). Thus, FLASH is robust for a range of reagent options and allows further optimization of the technique for specific antigens of interest.

[0137] To further test the effect of FLASH treatment on tissue morphology, FLASH-treated organs were embedded in paraffin and specimens previously imaged in three dimensions were analyzed by conventional 2D histology. Despite previous processing and analysis, the tissues were able to accept hematoxylin and eosin staining. Tissue structure remained intact, and different compartments such as blood vessels and ducts could be easily identified as in untreated controls (Fig. 8). Thus, FLASH enables deep tissue immunolabeling of intact organs while maintaining epithelial integrity and tissue structure.

[0138] [Example 2 - Imaging of the Adult Pancreatic Ductal System] Materials and Methods FLASH. FLASH was developed for the rapid detection of multiple antigens in intact adult organs by mild non-destructive epitope retrieval (rapid optical microscopy analysis of whole organs stained with FLASH-antibodies).

[0139] The mice were euthanized by cervical dislocation. Cardiac perfusion was performed with 20 ml of PBS. The pancreas attached to the spleen and duodenum was removed without disturbing the glands. The samples were fixed in 4% PFA overnight at 4°C. The specimens were washed twice with PBT (0.4% Triton X-100 in PBS (Sigma-Aldrich)) for 1 hour each and incubated overnight at 54°C in 200 mM boric acid (Sigma-Aldrich) and 4% SDS (Sigma-Aldrich) at pH 7.0.

[0140] The samples were washed with PBT for 3 hours with three volume exchanges. For immunolabeling, the samples were incubated in FLASH blocking buffer (1% bovine serum albumin (Sigma-Aldrich), 5% DMSO (Sigma-Aldrich), 10% fetal bovine serum (Gibco), 0.02% sodium azide (Sigma-Aldrich), 0.2% Triton X-100 in PBS) for 1 hour and incubated with the antisera (all 1:100) at room temperature for at least 16 hours on a nutator. The samples were washed with PBS by three volume exchanges and incubated with the secondary antibodies (all 1:100) at room temperature for at least 2 days.

[0141] The samples were washed with PBS by three volume exchanges and gradually dehydrated in 30%, 50%, 75%, 2×100% methanol (Sigma-Aldrich) for 1 hour each and immersed in methyl salicylate diluted with methanol in a glass dish: 25%, 50%, 75%, 2×100% methyl salicylate (Sigma-Aldrich) for 30 minutes each under light-shielded conditions.

[0142] The fluorescent proteins were stained by immunohistochemistry. The following antibodies were used: amylase (goat, SCBT), GFP (goat, Abcam), GFP (mouse, Roche), Krt19 TROMA III (rat, DSHB), Tomato (rabbit, Rockland). All secondary antibodies were Alexa-dye conjugates (ThermoFisher). The nuclei were stained with DRAQ5 (Biostatus).

[0143] Results To preserve the geometric complexity of the adult pancreas, we developed a new approach for rapid whole-organ three-dimensional immunostaining and imaging (FLASH, see Methods) that enables robust quantitative investigation of organ structure at single-cell and tissue levels. FLASH maintained pancreatic compartmentalization and tissue integrity (Figure 10a, b). The adult pancreatic ductal system was visualized by inducing tdTomato expression in all duct cells (R26-CAG-tdTomato; Hnf1b-CreERt2). FLASH imaging of whole pancreata revealed a complex hierarchy of ducts across exocrine lobules (Figure 9a, b, Figure 10c, d). Duct segments varied considerably in diameter (Figure 9c, d), and smaller ducts were composed of elongated cells and larger ducts of cuboidal cells (Figure 9, e, f). Confetti labeling showed mainly clonal expansion along the long cell axis (Figure 10e, f). These findings reveal the complexity and heterogeneity of the pancreatic ductal system (Figure 9g).

[0144] To induce epithelial transformation, conditional mosaic activation of the KrasG12D oncogene was induced by simultaneous deletion of either the p53 or Fbw7 tumor suppressor genes. FLASH analysis of KrasG12D;Fbw7 F / F;Ck19-CreERt (KFCk19) and KrasG12D;Fbw7 F / F;Hnf1β-CreERt (KFH) mice revealed that two morphologically distinct lesion types co-occurred in all pancreata analyzed. Transformed ducts either bulged basally away from the duct lumen (termed “outgrowth”) or invaginated apically towards the duct lumen (termed “ingrowth”) (Figures 11a and 12a–d). Outgrowth lesions elongated the duct lumen and formed spherical structures (Figures 11b, c, 12e–g), which progressed to back-to-back glandular duct neoplasms (Figure 11d). In contrast, ingrowth lesions grew papillarily into the duct lumen (Figures 11e, f, 12e) and progressed to intraductal neoplasms with local occlusion of the duct lumen (Figure 11g). Activation of KrasG12D and deletion of Fbw7 or p53 in acinar cells located at the tips of small ducts induced acinar-to-duct metaplasia (ADM), leading to Krt19-positive spherical lesions continuous with the duct tree (Figures 13a–f). Outgrowth and ingrowth lesions were also identified in mice with duct-specific (KPCk19) or whole pancreas (KPC) KrasG12D activation and p53 deletion, indicating that these observations are independent of specific oncogene combinations (Figures 14a–e).

[0145] [Example 3 - 3D Imaging of Human Tissue Biopsies] Materials and Methods FLASH. Human tissue biopsies were obtained from consenting patients with pancreatic ductal adenocarcinoma. Biopsies were fixed overnight in 10% NBF and either processed immediately by FLASH or embedded in paraffin for archival purposes. To perform FLASH on archival material, paraffin-embedded samples were first deparaffinized by incubation in HistoClear or xylene for 30 minutes, followed by washing with EtOH and sequential rehydration by washing in 90%, 75%, 30% EtOH, and subsequently 2×ddH2O for 30 minutes. Samples were incubated overnight at 54 °C in 200 mM boric acid (Sigma-Aldrich) and 8% Zwittergent® 3-10 (Merck) at pH 7.0.

[0146] Samples were washed with PBT for 3 hours with three volume exchanges. For immunolabeling, samples were incubated for 1 hour in FLASH blocking buffer (1% bovine serum albumin (Sigma-Aldrich), 5% DMSO (Sigma-Aldrich), 10% fetal bovine serum (Gibco), 0.02% sodium azide (Sigma-Aldrich), 0.2% Triton X-100 in PBS) and incubated with antisera (all 1:100) at room temperature for at least 16 hours on a nutator. Samples were washed with three volume exchanges of PBS and incubated with secondary antibodies (all 1:100) at room temperature for at least 2 days.

[0147] Samples were washed with PBS by three volume exchanges and gradually dehydrated in 30%, 50%, 75%, 2×100% methanol (Sigma-Aldrich) for 1 hour each, and immersed in methyl salicylate diluted with methanol in a glass dish: 25%, 50%, 75%, 2×100% methyl salicylate (Sigma-Aldrich) for 30 minutes each under light-shielded conditions.

[0148] The fluorescent protein was stained by immunohistochemistry. The following antibodies were used: Cdh1 (rat, Novex), Krt19 TROMA III (rat, DSHB), Muc5AC (rabbit, Atlas), SMA (mouse, Sigma-Aldrich). All secondary antibodies were Alexa-dye conjugates (ThermoFisher).

[0149] Results To test whether FLASH can be utilized, for example, in clinical histopathology for the analysis of human tissue samples, conventional biopsy analysis by standard histology on tissue sections was compared to FLASH-3D imaging of intact biopsies. Specifically, the optical 2D sections of the FLASH-imaged samples were compared to the 2D tissue sections of the standard-processed samples. It was found that FLASH enables immunolabeling and optical clearing of fresh, fixed, and paraffin-embedded biopsies. Exophytic and endophytic lesions were detected in samples cleared with FLASH (FIGS. 15a-d). FLASH immunolabeling identified ducts and mucus properties, as well as mesenchymal cells, in the biopsy samples in the spatial context of the surrounding normal and cancerous pancreatic tissue regions. The 2D optical sections of the 3D imaging dataset reproduced the presentation of tissues and lesion morphology in the standard histopathological analysis of tissue sections (FIGS. 15a, c). Thus, FLASH enables rapid tissue path characterization of human materials. All documents referred to herein are hereby incorporated by reference in their entirety, with particular attention to the subject inventions to which they are referred. Various modifications and changes to the described methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention.

[0150] Although the present invention has been described in connection with certain preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art of molecular biology, cellular immunology or related fields are within the scope of the following claims.

[0151] The present invention will now be described in more detail in the following numbered paragraphs: 1. A solution for preparing a tissue sample for three-dimensional (3D) imaging, comprising a buffer with a pH of less than 9 and a surfactant. 2. The solution according to paragraph 1, wherein the 3D imaging is based on immunostaining. 3. The solution according to paragraph 1 or paragraph 2, wherein the surfactant is SDS. 4. The solution according to paragraph 1 or paragraph 2, wherein the surfactant is an amphoteric surfactant. 5. The solution according to paragraph 4, wherein the amphoteric surfactant is a Zwittergent® surfactant. 6. The solution according to any one of paragraphs 1 to 5, wherein the pH of the buffer is about 7. 7. The solution according to any one of paragraphs 1 to 6, wherein the buffer is a borate or citrate buffer. 8. The solution according to any one of paragraphs 1 to 7, wherein the solution is used at a temperature between about 40°C and about 60°C. 9. The solution according to paragraph 8, wherein the solution is used at a temperature of about 55°C. 10. The solution according to any one of paragraphs 1 to 9, wherein the tissue sample is derived from a mouse, rat, rabbit, cow, pig or non-human primate. 11. The solution according to paragraph 10, wherein the tissue sample is derived from a human. 12. A method for preparing the solution according to any one of paragraphs 1 to 11, comprising combining the buffer with a pH of less than 8 with the surfactant. 13. A method for preparing a tissue sample for 3D imaging, comprising treating the tissue sample with the solution according to any one of paragraphs 1 to 11. 14. Use of the solution according to any one of paragraphs 1 to 11 for preparing a tissue sample for 3D imaging. 15. A kit for preparing a tissue sample for 3D imaging, comprising the solution according to any one of paragraphs 1 to 11.

Claims

1. A method for preparing a tissue sample for 3D imaging, comprising: a) treating the tissue sample with a solution containing a buffer with a pH less than 9 and a surfactant; and b) immunolabeling the tissue sample wherein the tissue sample is not embedded in a hydrogel and / or not fixed with glutaraldehyde.

2. The method according to claim 1, wherein the tissue sample is a surgically excised sample, a sample of 3D cell culture material (organoid), or a sample of tissue produced by biotechnology.

3. A method for preparing a tissue sample for 3D imaging, comprising: a) preparing a tissue sample; b) treating the tissue sample with a solution containing a buffer with a pH less than 9 and a surfactant; and c) immunolabeling the tissue sample wherein the tissue sample is not embedded in a hydrogel and / or not fixed with glutaraldehyde, and the tissue sample is a surgically excised sample, a sample of 3D cell culture material (organoid), or a sample of tissue produced by biotechnology.

4. The method according to any one of claims 1 to 3, wherein the 3D imaging is based on immunostaining.

5. a) the surfactant is SDS or zwitterionic; and / or b) the pH of the buffer is less than 8.5, less than 8, less than 7.5, or 7; and / or c) the buffer is a borate or citrate buffer The method according to any one of claims 1 to 4.

6. The surfactant is a Zwittergent (registered trademark) surfactant, and the Zwittergent (registered trademark) surfactant is selected from any one of n-octyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid, n-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid, n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid, n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid, and n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid. The method according to claim 5.

7. The buffer solution is boric acid. The method according to claim 5 or 6.

8. The solution is used at a temperature between about 40°C and about 60°C. The method according to any one of claims 1 to 7.

9. The solution is used at a temperature between about 50°C and about 60°C. The method according to claim 8.

10. The solution is used at a temperature of about 55°C or about 54°C. The method according to claim 9.

11. The tissue sample is a) a mouse, rat, rabbit, cow, pig or non-human primate; or b) a human Derived from. The method according to any one of claims 1 to 10.

12. The tissue sample is fixed using neutral buffered formalin. The method according to any one of claims 1 to 11.

13. The neutral buffered formalin is 10% neutral buffered formalin. The method according to claim 12.

14. Further comprising determining the presence or absence of a disease state in the tissue sample. The method according to any one of claims 1 to 13.

15. The method according to any one of claims 1 to 14, wherein the tissue sample is paraffin-embedded. **Claim 16** The method according to any one of claims 1 to 15, wherein the tissue sample is an intact tissue sample.

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