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

Abstract

To provide methods of preparing tissue samples for 3D imaging, which facilitate the use of a wider range of antibodies for immunolabeling of samples.SOLUTION: A method of preparing a tissue sample for 3D imaging is provided, the method comprising treating the tissue sample with a solution containing a buffer solution with pH below 9 and a detergent.SELECTED DRAWING: None

Description

[Technical Field]

[0001] 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. [Background technology]

[0002] Histology is the study of the anatomy of plant and animal cells and tissues using microscopy. It is typically studied using a light or electron microscope, with specimens sectioned, stained, and mounted on microscope slides. The ability to visualize or specifically identify microscopic structures is frequently enhanced through the use of stains. Histology is an essential tool in biology and medicine.

[0003] Histopathology is the 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 under brightfield microscopy to estimate various histological and pathological findings, such as tissue composition, tissue structure and morphology, cell morphology, cellular malignancy, degree of inflammation and fibrosis, depth of tumor infiltration, presence of tumor elements at the cut margins, and lymph node metastasis status.

[0004] Traditional methods of histology and histopathology involve staining and imaging using microscopy. However, while traditional methods provide information about morphological changes in cells and tissues, they also have fundamental limitations. For example, traditional methods can only provide flat, two-dimensional (2D) images, limiting their ability to observe three-dimensional (3D) structures consisting 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 typically evaluated. Therefore, concerns that additional significant lesions may exist in unassessed regions must still be addressed. Traditional 2D histological investigation of tissues limits adequate insight into 3D tissue structure. For example, 3D imaging is essential to explore the connectivity of local cellular networks.

[0005] However, preparing tissue samples for 3D imaging has proven challenging. Previous techniques for preparing tissue samples for 3D imaging focus on increasing tissue transparency to reduce the light-scattering properties of the multicellular layer and increase light transmittance, both of which are important for light microscopy. Previous techniques were developed for neural tissue. While their application also increases transparency in other tissues, they make it impossible to stain them, especially with immunolabeling.

[0006] Furthermore, previous techniques used in the art for preparing tissue samples for 3D imaging have utilized various enhanced fixation of the sample, such as hydrogel embedding, because such enhanced fixation was considered in the art to be necessary to achieve a tissue sample suitable for 3D imaging.

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

[0008] This is not possible with current methods in the art for preparing tissue samples for 3D imaging due to the limited number and types of antibodies that can be used with the resulting tissue samples.In this respect, the resulting tissue samples are not compatible with labeling by a wide range of antibody types and specificities.Without wishing to be bound by theory, this may be due to the masking of antigens during the enhanced fixation step. Summary of the Invention [Problem to be solved by the invention]

[0009] Thus, there is a need in the art for alternative methods of preparing tissue samples for 3D imaging that facilitate the use of, for example, a wider range of antibodies for immunolabeling of the 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 less complicated methods can be used to prepare tissue samples than those previously used in the art. This example demonstrates that no additional fixation, for example with hydrogel or glutaraldehyde (GA), is required to achieve a tissue sample suitable for 3D imaging by immunolabeling.

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

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

[0013] The present invention provides a solution comprising a buffer having a pH below 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.Therefore, advantageously, the present invention has general applicability in preparing tissue samples for 3D imaging.This example also demonstrates that the present invention can facilitate imaging with a resolution comparable to 2D histochemistry, and tissue integrity and organ structure are maintained.

[0015] The present invention is time- and cost-effective and does not require specialized equipment or sample procurement (e.g., hydrogel embedding, GA fixation, etc.), which represents a further advantage over techniques previously used in the art. As noted above, without wishing to be bound by theory, prior art techniques may have drawbacks associated with excessive fixation, complex epitope retrieval, and cross-linking that must be overcome to prepare tissue samples suitable for imaging by achieving tissue integrity, preserving organ architecture, and generating a reliable signal when immunolabeled.

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

[0017] The present invention advantageously facilitates the provision 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 across a wide range of tissues, and traditional histological techniques may not work with a wide range of antibodies, including antibodies of both broad and narrow specificity.

[0018] In another advantage, the present invention also allows for the imaging of complete, intact tissue samples without the need for serial sectioning of the sample. The present invention also advantageously allows for 3D imaging of partially sectioned or serially sectioned tissue samples to an extent that is consistent with or improves upon traditional histological techniques.

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

[0020] In traditional histology for pathology or diagnostic purposes, it is common to sample a portion of the whole tissue, and it is known that insufficient sampling can result in missing 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 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, which can advantageously reduce the likelihood that a disease state will go undetected.

[0021] In one advantage, the method or use of the present invention provides improved tissue samples for 3D imaging, which allows for a wider range of immunolabeling from tissue samples for 3D imaging.

[0022] As a further advantage, the present invention may also be used on archival tissue samples, for example those in paraffin blocks.

[0023] It is known that tissue samples archived (preserved) in paraffin blocks are contacted with and embedded in paraffin for preservation (paraffin embedding) using standard methods. It is also known that to retrieve the tissue sample from preservation, the preserved tissue sample is then deparaffinized and then processed for imaging. While conventional immunolabeling methods are not suitable for use with paraffin-embedded samples, the invention described herein is surprisingly suitable for use with such samples.

[0024] The solutions of the present 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 producing a tissue sample before it is subjected to 3D imaging.

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

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

[0027] The methods of the invention can be used to study healthy or diseased tissues or to investigate the effectiveness of candidate drugs in modifying disease. Solutions, kits and uses thereof useful in practicing the methods of the invention are also provided.

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

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

[0030] In another embodiment, the present invention provides a method of preparing a tissue sample for 3D imaging, comprising the steps of: a) preparing a tissue sample; b) fixing the tissue sample; c) archiving the tissue sample, if necessary; d) treating the tissue sample with a solution containing a buffer having a pH of less than 9 and a surfactant; e) optionally immunolabeling the tissue sample; The present invention provides a method comprising:

[0031] Advantageously, according to 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] Advantageously, according to the method of the present invention, 3D imaging may be based on immunostaining.

[0033] According to the method of the present invention, a) the detergent may be SDS or zwitterionic, preferably a Zwittergent® detergent, wherein the zwittergent detergent 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.

[0034] Suitably, in the method of the present invention, the solution may 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] Suitably, in accordance with the methods of the present invention, the tissue sample may be from: a) a mouse, rat, rabbit, cow, pig or non-human primate; or b) a human, preferably the tissue sample is a surgically excised specimen.

[0036] Suitably, according to 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, according to the method of the present invention, the tissue sample may be paraffin-embedded.

[0039] Suitably, according to 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 for preparing a tissue sample for three-dimensional (3D) imaging, comprising a buffer having a pH below 9 and a surfactant.

[0041] Advantageously, said 3D imaging may be based on immunostaining.

[0042] Suitably, a) the detergent may be SDS or zwitterionic, preferably a Zwittergent® detergent, wherein the zwittergent detergent 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] Suitably, 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] Suitably, depending on the solution, the tissue sample may be from: a) a mouse, rat, rabbit, cow, pig or non-human primate; or b) a human, preferably the tissue sample is a surgically excised specimen.

[0045] In one embodiment, the invention provides a method of preparing a solution according to the invention, comprising combining said buffer having a pH below 8 with said 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 for preparing a tissue sample for three-dimensional (3D) imaging, comprising a buffer having a pH below 9 and a surfactant, wherein 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 citrate.

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

[0052] Preferably, the surfactant is zwitterionic.

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

[0054] Preferably, the surfactant is SDS.

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

[0056] In one embodiment, the present invention provides a solution for preparing a tissue sample for three-dimensional (3D) imaging, comprising a buffer having a pH less than 8.5 and a surfactant, wherein the surfactant is SDS and the buffer is a boric acid solution. The present invention also relates to the following: [Item 1] 1. A method of preparing a tissue sample for 3D imaging, comprising treating the tissue sample with a solution comprising a buffer having a pH below 9 and a surfactant. [Item 2] 1. A method of 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 below 9 and a surfactant; and e) immunolabeling said tissue sample A method comprising: [Item 3] 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] 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 a 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 borate or citrate buffer, preferably borate; The method according to any one of items 1 to 4. [Item 6] 6. The method according to any one of items 1 to 5, wherein the solution is used at a temperature of about 40°C to about 60°C, preferably about 50°C to about 60°C, more preferably about 55°C or about 54°C. [Item 7] The tissue sample a) a mouse, rat, rabbit, cow, pig, or non-human primate; or b) Humans 7. The method according to any one of items 1 to 6, wherein the tissue sample is derived from a surgically excised sample, a sample of 3D cell culture material (organoid) or a sample of bioengineered tissue. [Item 8] 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] 9. The method of any one of items 1 to 8, further comprising determining the presence or absence of a disease state in the tissue sample. [Item 10] 10. The method according to any one of items 1 to 9, wherein the tissue sample is paraffin-embedded. [Item 11] 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 tissue samples for three-dimensional (3D) imaging, comprising a buffer having a pH below 9 and a surfactant. [Item 13] 13. The solution according to item 12, wherein the 3D imaging is based on immunostaining. [Item 14] a) the surfactant is SDS or a 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 borate or citrate buffer, preferably borate; Item 14. The solution according to item 12 or 13. [Item 15] 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 a) a mouse, rat, rabbit, cow, pig, or non-human primate; or b) Humans 16. The solution according to any one of items 12 to 15, wherein the tissue sample is derived from a surgically resected specimen, a sample of 3D cell culture material (organoid) or a sample of bioengineered tissue. [Item 17] 17. A method for preparing the solution according to any one of items 12 to 16, comprising combining the buffer solution having a pH of less than 8 with the surfactant. [Item 18] 17. Use of the solution according to any one of items 12 to 16 for preparing a tissue sample for 3D imaging. [Item 19] 19. Use of the solution according to item 18 for determining the presence or absence of a disease state. [Item 20] 17. A kit for preparing tissue samples for 3D imaging, comprising the solution according to any one of items 12 to 16. [Brief explanation of the drawings]

[0057] [Figure 1] Figure 1: Epitope retrieval enables whole tissue immunolabeling. a) Intact pancreatic lobes were treated with the indicated buffer containing 4% SDS for 16 hours at the indicated temperature. Staining of Krt19 (pancreatic duct) 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. Crosses indicate sample loss. [Figure 2]Figure 1: Tissue clearing is not sufficient for immunolabeling. a) Immunofluorescence 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 fields are shown. Scale bar 100 μm. c) 3D reconstruction of pancreas stained with the indicated antibodies 16 hours after FLASH processing. [Figure 3] FLASH enables 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) Enlargement of the area (white box) indicated in (a) showing the complex arrangement of myoepithelial cells around the bronchiolar tubes. Scale bar 500 μm. c) Enlargement of the area (white box) indicated in (b). Scale bar 150 μm. c') Optical section demonstrating intact compartmentalization of the epithelial and myoepithelial tissue layers. Scale bar 50 μm. d) Comparative staining of a paraffin-embedded lung tissue section (4 μm). Scale bar 50 μm. e) Staining for CC10, SMA, and Sp-C (alveolar type II cells). Scale bar 100 μm. [Figure 4] FLASH enables 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 a bile duct stained for Krt19 (ductular cells) and Aqp1 (microcirculation). Scale bar 200 μm. c) Staining for Krt19 and Prox1 (lymphatic endothelium, nuclei). Scale bar 100 μm. d) Bile duct stained for DBA (ductular cells) and CD44 (cholangiocytes). Scale bar 50 μm. d') Optical section of the duct shown in (d) demonstrating maintained localization of CD44 to the lateral plasma membrane. Scale bar 20 μm. [Figure 5]FLASH enables immunostaining of the lacrimal gland. a) 3D reconstruction of the lacrimal gland stained for Krt19 (duct cells), SMA (stroma), and Aqp1 (microcirculation). 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 compartmentalization of the mesenchymal and epithelial tissue layers. Scale bar 100 μm. [Figure 6] FLASH enables immunostaining of the kidney. a) 3D reconstruction of a 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 (glomeruli, nuclei). Scale bar 100 μm. b') Optical section through the indicated region (white box) in (b). Scale bar 50 μm. c) Staining for DBA, PNA, and WT1 on a paraffin-embedded kidney section (4 μm). Scale bar 50 μm. [Figure 7] Figure 1: Preserved subcellular protein localization after FLASH. a) 3D image of a FLASH-processed 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 region 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 in (a) showing the different subcellular localization of the epitopes. c) Single-channel image of (a'). d) Staining for Amy, CollIV, and Mist1 on a paraffin-embedded pancreatic tissue section (4 μm). Scale bar 20 μm. e) Intensity profile of the fluorophores in (d). [Figure 8]Figure 1 shows 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 preserved tissue morphology and the suitability of using FLASH-treated samples for subsequent 2D staining and histological analysis. All scale bars 100 μm. [Figure 9-1] FLASH imaging of the intact pancreas and visualization of the ductal tree. (a-c) 3D rendering of a tdTomato-stained intact pancreas from a tamoxifen-treated R26-CAG-tdTomato;Hnf1βCreERT2 mouse. (a) 3D image of the entire pancreas attached to the duodenum and spleen. Scale bar: 5 mm. (b) 3D enlargement of the area in (a). Scale bar: 500 μm. (c) 3D image of ductal segments. (1) Main duct; (2) Interlobular duct; (3) Intralobular duct; (4) Intervening duct. Scale bar: 100 μm. (d) Segmental heterogeneity of ductal diameter. Each dot represents one duct, 180 ducts, 3 mice. (e) Pancreatic tissue section stained for Cdh1 and DNA to highlight different ductal cell shapes. Scale bar: 10 μm. (f) Segmental heterogeneity of duct cell width (black), height (red), and length (blue); average of 5 cells per duct, 115 ducts, 6 mice. The fitted line was obtained using nonlinear regression. (g) Illustration of segmental heterogeneity of the duct tree. L - cell length, W - cell width, H - cell height. [Figure 9-2] This is a continuation of Figure 9-1. [Figure 9-3] This is a continuation of Figure 9-2. [Figure 10-1]Figure 1: Preserved organ integrity after FLASH. (a) FLASH staining of an insulin (Ins)-GFP reporter mouse pancreas for amylase (Amy), Krt19, and Ins-GFP. Left: 3D reconstruction demonstrating the complex organization of islets and pancreatic ducts. Scale bar: 100 μm. Right: Optical section through the indicated area (left) showing 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 area of (b) demonstrating a continuous ductal cell monolayer and preserved ductal lumen. Scale bar: 30 μm. Right: Staining for Krt19 and DNA on a 4 μm histological section of a 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) intraperitoneal injection of 100 μg tamoxifen per gram of body weight. Scale bar: 500 μm. (d) Arborization of the ductal tree. Each dot represents one duct, and lines indicate branches. Z-stacks of at least 30 random high-magnification images were taken for three Krt19-stained pancreases. To classify the mode of arborization, the largest duct per image was identified, and the diameters of the four subsequent branching ducts were measured. For ducts with multiple branches, the sequence was continued with the largest duct branching from it. Terminal duct cells were assigned a duct diameter of 0 μm to indicate the end of the ductal tree. (e) Cell division direction was measured as the angle determined relative to a line connecting the nucleus and tube orientation in two-cell clones from tamoxifen-treated R26-LSL-Confetti;Hnf1β-CreERt2 mice (263 clones, 5 mice). (f) Cell division direction and aspect ratio in tubes of various diameters. Solid lines represent exponential fits. [Figure 10-2] This is a continuation of Figure 10-1. [Figure 11]Heterogeneity of neoplasia induction in pancreatic ducts. (a) Exophytic and endophytic pancreatic ductal transformations. (b-g) FLASH comparison with 2D histology. (b, e) 3D images (left) and optical sections (right) of exophytic (b) and endophytic (e) transformations 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 exophytic (c-d) and endophytic (f-g) foci 10 days (c, f) and 21 days (d, g) after recombination. Scale bar: 100 μm. [Figure 12-1]Figure 1 shows exophytic and endophytic neoplasia 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 transformed cell were quantified in KFCk19 mice 1 week after tamoxifen injection. EYFP-tracked Krt19+ cells per duct were quantified, and the total number of ductal cells was estimated by dividing the duct length by the average cell length measured for that duct and multiplying this by the average number of cells circumscribing that duct. Transformed clones were recognized as groups of more than three EYFP-tracked cells sharing an interface. Each dot represents one duct (112 ducts, 3 mice). (c-d) KFH mouse model for alternative targeting of the pancreatic duct. (d) Left, 3D rendering of an exophytic neoplasm (top) and an endophytic neoplasm (bottom) in a KFH mouse. Stained for Krt19 and tdTomato. Scale bar: 100 μm. Right, H&E staining for exophytic (top) and endophytic (bottom) foci in KFH. Scale bar: 100 μm. (e) H&E and AB / PAS staining for exophytic and endophytic foci in a KFCk19 mouse, demonstrating the nonmucinous nature common in neoplasms of ductal origin. Scale bar: 100 μm. (f) Experimental strategy for visualizing the relationship of exophytic neoplasms to the ductal tree. The extrahepatic bile duct was cannulated at the ampulla, and the pancreatic ductal tree was perfused with 50 μl of FITC-labeled dextran. (g) DexFITC uptake by an exophytic KFCk19 foci, demonstrating the foci's relationship to the ductal system. Left, 3D image, and right, optical section. Scale bar 50 μm. [Figure 12-2] This is a continuation of Figure 12-1. [Figure 12-3] This is a continuation of Figure 12-2. [Figure 13-1]Morphological progression of acinar-derived neoplasia. (a) Schematic illustrating the genetic strategy for acinar cell transformation by KrasG12D activation with 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 metaplasia identified by focal upregulation of Krt19 expression in acinar cells. td-Tomato-tracked acini are shown connected to terminal ducts. Arrowheads demarcate Krt19 expression by the central acini, forming a small ring of acinar-derived td-Tomato-tracked Krt19+ cells. Scale bar: 50 μm. (c) 3D projection of a spherical KFEla1 foci contacting a small duct (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 mouse (KPEla1). (d) 3D image of a spherical td-Tomato-traced KPEla1 foci connected to distal ducts (arrowheads). Scale bar: 50 μm. (e) 3D projection of a large KPEla1 foci showing the central grape-like morphology of the back-to-back spherical structures and maintained connection to several small-diameter ducts (arrowheads) at the foci's edge. Scale bar: 200 μm. (e') Higher magnification of the indicated area in (e), demonstrating seamless connection between acinar-derived Krt19+ cells and wild-type ductal epithelium (dotted line). Scale bar: 30 μm. (f) Retrograde perfusion of the ductal tree with dextran-FITC as in Extended Data Fig. 2f demonstrates direct connection of acinar-derived foci to the ductal system. 3D image of KPEla1 foci. Scale bar 50 μm. (g) H&E staining of KPEla1 foci demonstrating spherical morphology. Scale bar 100 μm. (h) KrasG12D;p53 F / F;Ptf1a-CreERt2(KPPtf1a) mouse. H&E staining demonstrating spherical morphology of foci. Scale bar 100 μm. [Figure 13-2] This is a continuation of Figure 13-1. [Figure 14-1]Figures showing exophytic and endophytic neoplasia in the KrasG12D;p53 F / F model and human pancreas. (a-c) Endophytic and exophytic foci induced by p53 deletion with KrasG12D activation after Pdx1-Cre-induced pancreatic recombination (KPC). (b) 3D images of pancreatic regions with endophytic (1) and exophytic (2) transformation. Scale bar: 150 μm. (1-2) Higher magnification of the area indicated in (b). Scale bar: 50 μm. (1) The arrowhead defines a common invagination in the endophytic growth. (2) The dotted line marks a morphologically normal small-diameter duct in contact with a spherical exophytic foci. (c) Hematoxylin and eosin (H&E) staining for exophytic (left) and endophytic (right) foci in the KPC model. Scale bar: 100 μm. (d-e) Exophytic and endophytic foci induced by p53 deletion with KrasG12D activation in the pancreatic duct (Ck19-CreERt;KPCk19). (e) 3D projection of exophytic (left) and endophytic (right) foci shapes in KPCk19 mice. Scale bar 100 μm. (f) H&E staining of tissue sections from background pancreas of a patient presenting with pancreatic ductal adenocarcinoma. (Left) Exophytic foci. (Right) Endophytic foci. Scale bar 100 μm. [Figure 14-2] This is a continuation of Figure 14-1. [Figure 15] Figure 1 shows FLASH 3D imaging of human biopsies. (a-b) Exophytic foci in the human pancreas. (a) H&E staining of a tissue section from a human pancreas. Scale bar 100 μm. (b) A human pancreatic biopsy imaged with FLASH showing exophytic ductal foci identified by Krt19 immunolabeling. Left, 3D image. Right, optical section. (c-d) Endophytic foci. (c) H&E staining of a human pancreas. Scale bar 100 μm. (d) A human pancreatic biopsy imaged with FLASH showing endophytic ductal foci 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. DETAILED DESCRIPTION OF THE INVENTION

[0058] 3D imaging The present invention allows visualization of molecularly labeled structures within large intact tissues in three dimensions.

[0059] 3D imaging can be performed by methods known to those skilled in the art, for example, by optical microscopy, fluorescence microscopy, such as confocal microscopy, light sheet microscopy, super-resolution microscopy, spectral precision distance microscopy, activated luminescence elimination, magnification microscopy or optical projection tomography and any variants thereof.

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

[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 retrieval without disrupting tissue architecture. Unlike previous techniques, 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 to detect specific proteins in a sample, for example, immunolabeling or immunohistochemical staining of tissue sections.

[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 immunofluorescence labeling, which is cell staining) is perhaps the most commonly applied immunostaining technique. While immunolabeling and immunofluorescence labeling use fluorescent dyes, immunohistochemistry and immunocytochemistry use enzymes, such as peroxidase and alkaline phosphatase. These enzymes can catalyze reactions that produce colored products that are easily detectable by light microscopy. Alternatively, radioactive elements can be used as labels, and the immunoreaction can be visualized by autoradiography.

[0064] Several methods can be used for immunostaining specific antigens. 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 then the antigen bound to the primary antibody is stained (secondary antibody method). In yet another method, a label can be prepared by directly binding a fluorescent label and biotin, and then the antigen bound to the primary antibody and a secondary antibody modified with avidin or streptavidin is stained (biotin-avidin method or sandwich method).

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

[0066] In one aspect, the antigen may 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] Additionally, any secondary antibody can be used, which will vary depending on the primary antibody, examples of which include anti-mouse, rabbit, bovine, goat, sheep, dog, and chicken antibodies.

[0068] Any known method can be used for binding of fluorescent labels to antibodies or biotin, such as amidation by reaction between an amine and a carboxylic acid, sulfidation 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.

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

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

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

[0072] buffer solution 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. The pH of a buffer changes only slightly when a small amount of a strong acid or base is added to it. Buffer solutions are used as a means of maintaining a nearly constant pH in a variety of applications.

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

[0074] In one embodiment, the buffer is selected from the group consisting of citric acid, acetic acid, borate, CHES, KH2PO4, Na2HPO4, TAPS ([tris(hydroxymethyl)methylamino]propanesulfonic 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), The buffer may be selected from the group consisting of 4-(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 (dimethylarsenate), MES (2-(N-morpholino)ethanesulfonic acid), and combinations thereof. In one aspect, the buffer may be phosphate buffered saline (PBS).

[0075] In a preferred embodiment, the buffer is a borate or citrate buffer solution.

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

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

[0078] In one embodiment, 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 embodiment, the pH of the buffer is about 7.0.

[0080] temperature In one embodiment, the solution according to the present invention is intended to be or should be used at a temperature between about 30°C and about 100°C. For example, the solution may be used at a temperature between about 40°C and about 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 embodiment 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 may 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] surfactants Suitable surfactants for use in the present invention are those surfactants used in standard methods of tissue sample preparation.

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

[0083] Nonionic surfactants include BigCHAP (N,N-bis[3-(D-gluconamido)propyl]cholamide), 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 (octyl phenol), and the like. The polyoxyethylene glycol sorbitan monooleate may be selected from the group consisting of octyloxypoly(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 embodiment of the present invention, the surfactant is an ionic surfactant. The ionic surfactant may 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 embodiment, the surfactant is a zwitterionic surfactant. The polar head group of a zwitterionic surfactant contains both negatively and positively charged groups, thus resulting in an overall neutral charge. The strength of action of these compounds is considered to be intermediate between ionic and nonionic surfactants, and they share characteristics of both types.

[0086] Zwitterionic detergents may 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 embodiment, the zwitterionic surfactant is a Zwittergent® surfactant, which may 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 invention, the surfactant is Zwittergent® 3-10 (n-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid).

[0089] Zwittergent® surfactants are commercially available, for example, from Calbiochem (Merck KGaA, Damstadt, Germany).

[0090] The amount of surfactant to include in a solution according to the present invention can be determined by one skilled in the art.

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

[0092] additives To further enhance staining, compounds that aid antigen retrieval or increase sample penetration can be added to the solution. Compounds that support antigen retrieval can be, for example, formaldehyde scavengers such as ascorbic acid, urea, Tris, 2-imidazolidinone, catalysts such as anthranilic acid and phosphanilates, or proteases such as trypsin, 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, β-mercaptoethanol, and tris(2-carboxyethyl)phosphine hydrochloride.

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

[0094] The tissue sample may be selected from epithelial, connective, muscle and nervous tissue.

[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, mammary tissue, head and neck tissue, intestinal tissue, salivary gland tissue, nerve tissue, ovarian tissue, testicular tissue, muscle tissue, and skin tissue.

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

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

[0098] In one embodiment, the tissue sample may be a tumor sample. The tumor may be derived from squamous cell carcinoma or carcinoma, lung cancer, cancer of the peritoneum, 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 or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, or head and neck cancer.

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

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

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

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

[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 quick freezing on dry ice or liquid nitrogen.

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

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

[0108] method The solutions according to the invention described herein may be used in methods for preparing tissue samples for 3D imaging.

[0109] Methods according to the present 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, including, but not limited to, cancer, autoimmune diseases, inflammatory diseases, metabolic diseases, neurodegenerative diseases, endocrine / reproductive system diseases, cardiovascular / pulmonary diseases, musculoskeletal diseases, or gastrointestinal diseases.

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

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

[0113] It is envisaged that the method according to the invention may be used as a step in determining whether a patient is or has responded to a therapeutic treatment by preparing a tissue sample for 3D imaging using a solution according to the invention.

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

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

[0116] The solutions of the present invention can be used to clear, ie, to make tissue samples transparent.

[0117] The method according to the invention may optionally comprise additional steps.

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

[0119] The method may include any one or more of the following steps: 1. Remove the tissue and, if necessary, perform vascular perfusion with PBS. 2. Tissue fixation. 3. Long-term tissue preservation as needed. 4. Washing step. 5. If applicable, clear the sample of debris and adjacent tissue. If necessary, the sample may be cut into smaller pieces. 6. Tissue incubation in a solution that allows immunolabeling. 7. Incubation with primary or secondary antibody.

[0120] In one aspect, the method comprises incubating a tissue sample with a solution according to the present invention at a temperature between about 30°C and about 100°C. For example, incubation may be 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 invention, incubation may be at a temperature between about 50°C and 60°C, preferably about 55°C. Incubation may 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, incubation may be at a temperature of about 54 or 55°C.

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

[0122] In one aspect of the present invention, the solution can be used to prepare tissue samples for 3D imaging before processing the sample for 2D staining.For example, tissue samples can undergo 3D imaging before processing for 2D imaging.In one aspect, tissue samples 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 aspect of the invention, the solution may be used in a method of preparing tissue samples for 3D imaging for analysis of human patient material in the clinic.

[0124] In another embodiment of the invention, the solution may be used to prepare tissue samples for 3D imaging for veterinary medicine.

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

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

[0127] The kit may also include components to facilitate 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 aid those skilled in the art in carrying out the present invention and are not intended to limit the scope of the invention in any way.

[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 (FLASH—rapid light microscopic analysis of antibody-stained intact organs).

[0130] Mice were euthanized by cervical dislocation. Cardiac perfusion was performed with 20 ml of PBS. Organs were harvested and fixed in 10% NBF overnight at 4°C. Specimens were washed twice for 1 hour with PBT (0.4% Triton X-100 (Sigma-Aldrich) in PBS). For the buffer comparison in Figure 1, samples were incubated for 16 hours in a solution of the indicated composition containing 4% SDS. For FLASH staining of various organs shown in Figures 2–5, samples were incubated for 16 hours at 54°C 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 for 24 hours at 54°C 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] Samples were washed with PBT for over 3 hours, with at least three volume changes. For immunolabeling, 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), and 0.2% Triton X-100 in PBS) for 1 hour and then incubated with antisera (all 1:100) on a nutator for at least 16 hours at room temperature. Samples were washed with three volume changes of PBS and incubated with secondary antibodies (all 1:100) for at least 2 days at room temperature.

[0132] The samples were washed with PBS three times with volume changes, gradually dehydrated in 30%, 50%, 75%, and 2 × 100% MeOH (Sigma-Aldrich) for 1 h each, and then immersed in glass dishes in methyl salicylate diluted in MeOH: 25%, 50%, 75%, and 2 × 100% MeOH (Sigma-Aldrich) for 30 min each, protected from light.

[0133] Fluorescent proteins were stained by immunofluorescence using the following antibodies: 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), and WT-1 (rabbit, SCBT). All secondary antibodies were Alexa dye conjugated (ThermoFisher). Nuclei were stained with DRAQ5 (Biostatus). The following lectins were used: DBA-FITC (VectorLabs), DBA-rhodamine (VectorLabs), and PNA-FITC (VectorLabs).

[0134] result To enable immunolabeling of intact tissue, we developed a combination of tissue permeabilization and partial reversal of protein crosslinks to restore antigenicity without compromising tissue integrity. A range of different buffer systems was tested in combination with membrane solubilization and low heat, followed by antibody incubation in a solvent-rich blocking reagent. The fragile, highly compartmentalized structure of the pancreas is inherently difficult to process for histology because it is prone to injury from the digestive enzyme-rich pancreatic juice. After incubation in buffer with a single overnight antigen retrieval step, whole pancreatic lobules were processed to label pancreatic ducts. Remarkably, all buffers tested yielded robust staining of branching ducts throughout the pancreatic lobule. While mild heat was required to achieve staining, higher temperatures destabilized tissue integrity, causing sample loss (Figure 1). Intact pancreata were then processed with FLASH followed by staining with amylase, Pcsk1, and Sma antibodies, which had previously failed with conventionally cleared pancreata. All stainings produced robust signals detectable in three dimensions using standard confocal microscopy, consistent with the antibody's performance in traditional 2D staining (Figure 2).

[0135] FLASH was successfully applied to the lung, liver, and lacrimal gland without further technical adaptation, resulting in a three-dimensional labeling distribution that was highly consistent with that seen in 2D tissue staining (Figures 3-5). The range of antibodies supported allowed visualization of the structure of all tissue components, sometimes simultaneously, including ducts, vasculature, interstitium, innervation, and lymphatic systems, as well as tissue-specific cell types such as hepatocytes, pancreatic acinar, and alveolar cells (Figures 3-5).

[0136] To further expand the range of compatible staining reagents to antibodies that may be sensitive to residual SDS in FLASH-processed samples, alternative membrane-permeable detergents were tested. The zwitterionic detergent Zwittergent-3-10 (FLASH Reagent 2) produced robust immunostaining in all tissues analyzed and improved performance of challenging antibodies, such as those against the transcription factors WT1 and Mist1. Importantly, tissue and cellular integrity was preserved, and subcellular staining distribution correlated with traditional 2D immunofluorescence as before (Figures 6-7). Thus, FLASH is robust across a range of reagent options, allowing for further optimization of the technique for specific antigens of interest.

[0137] To further examine 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 receptive to hematoxylin and eosin staining. Tissue architecture remained intact, and different compartments, such as blood vessels and ducts, could be easily identified, as in untreated controls (Figure 8). Thus, FLASH allows for deep-tissue immunolabeling of intact internal organs while preserving epithelial integrity and tissue architecture.

[0138] Example 2 - Imaging of the adult pancreatic duct system Materials and Methods FLASH. FLASH was developed for the rapid detection of multiple antigens in intact adult organs by light, non-destructive epitope retrieval (FLASH—rapid light microscopic analysis of antibody-stained intact organs).

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

[0140] Samples were washed with three volume changes of PBT for 3 hours. For immunolabeling, 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), and 0.2% Triton X-100 in PBS) for 1 hour and then incubated with antisera (all 1:100) on a nutator for at least 16 hours at room temperature. Samples were washed with three volume changes of PBS and incubated with secondary antibodies (all 1:100) at room temperature for at least 2 days.

[0141] The samples were washed with PBS three times with volume changes, gradually dehydrated in 30%, 50%, 75%, and 2 × 100% MeOH (Sigma-Aldrich) for 1 h each, and then immersed in methyl salicylate diluted in MeOH in glass dishes: 25%, 50%, 75%, and 2 × 100% methyl salicylate (Sigma-Aldrich) for 30 min each, protected from light.

[0142] 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), and Tomato (rabbit, Rockland). All secondary antibodies were Alexa-dye conjugated (ThermoFisher). Nuclei were stained with DRAQ5 (Biostatus).

[0143] result To preserve the geometric complexity of the adult pancreas, we developed a novel method for rapid whole-organ 3D immunostaining and imaging (FLASH, see Methods), which allows for robust quantitative investigation of organ architecture at the single-cell and tissue levels. FLASH maintained the compartmentalization and tissue integrity of the pancreas (Figure 10a, b). We visualized the adult pancreatic ductal system by inducing tdTomato expression in all ductal cells (R26-CAG-tdTomato; Hnf1b-CreERt2). FLASH imaging of the complete pancreas revealed a complex hierarchy of tubules throughout the exocrine lobule (Figure 9a, b; Figure 10c, d). Duct segments varied considerably in diameter (Figure 9c, d), with smaller ducts composed of elongated and cuboidal cells (Figure 9e, f). Confetti labeling demonstrated clonal expansion primarily along the long cell axis (Figure 10e, f). These findings reveal the complexity and heterogeneity of the pancreatic ductal system (Fig. 9g).

[0144] To induce epithelial transformation, we induced conditional mosaic activation of the KrasG12D oncogene 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 occurred simultaneously in all pancreatic tissues analyzed. Transformed ducts either bulged basally away from the ductal lumen (termed "exophytic") or invaginated apically toward the ductal lumen (termed "endophytic") (Figure 11a and Figure 12a-d). Exophytic foci extended the ductal lumen, forming spherical structures (Figure 11b, c, Figure 12e-g), which progressed to back-to-back adenoid ductal neoplasms (Figure 11d). In contrast, endophytic foci grew papillarily into the ductal lumen (Figures 11e, f, 12e) and progressed to intraductal neoplasia with focal obstruction of the ductal lumen (Figure 11g). Activation of KrasG12D and deletion of Fbw7 or p53 in acinar cells located at the tip of small ducts induced acinar-to-ductal metaplasia (ADM) and led to Krt19-positive spherical foci continuous with the ductal tree (Figures 13a-f). Exophytic and endophytic foci were also identified in mice with duct-specific (KPCk19) or pancreatic (KPC) KrasG12D activation and p53 deletion, indicating that these observations are independent of the specific oncogene combination (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 immediately processed for 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 min, followed by a wash in EtOH and stepwise rehydration with 30-min washes in 90%, 75%, and 30% EtOH, followed by 2× ddH2O. Specimens were incubated overnight at 54°C in 200 mM boric acid (Sigma-Aldrich) and 8% Zwittergent® 3-10 (Merck), pH 7.0.

[0146] Samples were washed with three volume changes of PBT for 3 hours. For immunolabeling, 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), and 0.2% Triton X-100 in PBS) for 1 hour and then incubated with antisera (all 1:100) on a nutator for at least 16 hours at room temperature. Samples were washed with three volume changes of PBS and incubated with secondary antibodies (all 1:100) at room temperature for at least 2 days.

[0147] The samples were washed with PBS three times with volume changes, gradually dehydrated in 30%, 50%, 75%, and 2 × 100% MeOH (Sigma-Aldrich) for 1 h each, and then immersed in methyl salicylate diluted in MeOH in glass dishes: 25%, 50%, 75%, and 2 × 100% methyl salicylate (Sigma-Aldrich) for 30 min each, protected from light.

[0148] Fluorescent proteins were stained by immunohistochemistry using the following antibodies: Cdh1 (rat, Novex), Krt19 TROMA III (rat, DSHB), Muc5AC (rabbit, Atlas), and SMA (mouse, Sigma-Aldrich). All secondary antibodies were Alexa-dye conjugated (ThermoFisher).

[0149] result To test whether FLASH can be utilized for the analysis of human tissue samples, e.g., in clinical histopathology, we compared conventional biopsy analysis by standard histology on tissue sections with FLASH-3D imaging of intact biopsies. Specifically, we compared optical 2D sections of FLASH-imaged samples with 2D tissue sections of standard-processed samples. We found that FLASH enables immunolabeling and optical clearing of fresh, fixed, paraffin-embedded biopsies. Exophytic and endophytic lesions were detected in FLASH-cleared samples (Figure 15a-d). FLASH immunolabeling identified ductal and mucinous features, as well as mesenchymal cells, in the biopsy samples within the spatial context of surrounding normal and cancerous pancreatic tissue regions. 2D optical sections of the 3D imaging dataset reproduced the presentation of tissue and lesion morphology in standard histopathological analysis of tissue sections (Figure 15a, c). Thus, FLASH enables rapid histopathological characterization of human material. All documents referred to in this specification are hereby incorporated by reference in their entirety, with particular attention being paid to the subject invention to which they refer. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention.

[0150] Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed 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 molecular biology, cellular immunology, or related fields are intended to be within the scope of the following claims.

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

Claims

1. 1. A method of preparing a tissue sample for 3D imaging, comprising treating the tissue sample with a solution comprising a buffer having a pH below 9 and a surfactant.

2. 1. A method of 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 below 9 and a surfactant; and e) immunolabeling said tissue sample A method comprising:

3. 3. The method of claim 1 or 2, wherein the tissue sample is not embedded in a hydrogel and / or is not fixed with glutaraldehyde.

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 a 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 borate or citrate buffer, preferably borate; The method according to any one of claims 1 to 4.

6. 6. The method according to any one of claims 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 at a temperature of about 55°C or about 54°C.

7. The tissue sample a) a mouse, rat, rabbit, cow, pig, or non-human primate; or b) Humans 7. The method according to any one of claims 1 to 6, wherein the tissue sample is derived from a surgically excised sample, a sample of 3D cell culture material (organoid) or a sample of bioengineered tissue.

8. The method of any one of claims 1 to 7, wherein the tissue sample is fixed using neutral buffered formalin, preferably 10% neutral buffered formalin.

9. 9. The method of any one of claims 1 to 8, further comprising determining the presence or absence of a disease state in the tissue sample.

10. The method of any one of claims 1 to 9, wherein the tissue sample is paraffin-embedded.

11. The method of any one of claims 1 to 10, wherein the tissue sample is an intact tissue sample.

12. A solution for preparing tissue samples for three-dimensional (3D) imaging, comprising a buffer having a pH below 9 and a surfactant.

13. The solution of claim 12, wherein the 3D imaging is based on immunostaining.

14. a) the surfactant is SDS or a 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 borate or citrate buffer, preferably borate; 14. The solution of claim 12 or claim 13.

15. 15. The solution according to any one of claims 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.

16. The tissue sample a) a mouse, rat, rabbit, cow, pig, or non-human primate; or b) Humans 16. The solution according to any one of claims 12 to 15, wherein the tissue sample is derived from a surgically excised specimen, a sample of 3D cell culture material (organoid) or a sample of bioengineered tissue.

17. 17. A method of preparing the solution of any one of claims 12 to 16, comprising combining the buffer with a pH of less than 8 with the surfactant.

18. Use of a solution according to any one of claims 12 to 16 for preparing tissue samples for 3D imaging.

19. 20. Use of the solution of claim 18 to determine the presence or absence of a disease state.

20. A kit for the preparation of tissue samples for 3D imaging, comprising a solution according to any one of claims 12 to 16.

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