A three-dimensional imaging method for biological tissue samples based on CUBIC clearing antibody staining

Abstract

This invention discloses a three-dimensional imaging method for biological tissue samples based on CUBIC clearing antibody staining, comprising: placing mouse brain tissue in CUBIC-1 solution for degreasing and dehydration; subjecting the degreased and dehydrated mouse brain tissue to sequential cell membrane permeation and blocking treatments; incubating the blocked mouse brain tissue with diluted primary antibody GFAP and secondary antibody Alexa488; washing the mouse brain tissue after two antibody incubations; placing the washed mouse brain tissue in CUBIC-2 solution for matching, and then embedding the mouse brain tissue in resin; placing the resin-embedded sample in a buffer bath, and acquiring images using a TDI-fMOST camera. This invention achieves overall staining of large biological tissue samples using CUBIC clearing antibody staining.

Description

Technical Field

[0001] This invention relates to the field of whole-body staining and three-dimensional imaging technology for biological tissue samples, specifically to a three-dimensional imaging method for biological tissue samples based on CUBIC clearing antibody staining. Background Technology

[0002] In routine biomedical research, the ideal attributes of tissue clearing techniques are safety, ease of operation, scalability, low environmental burden, and high transparency. Hydrophilic tissue clearing methods have the potential to meet all these requirements. CUBIC reagents based on hydrophilic agents have been developed to prioritize safety and reduce environmental burden, and these protocols only require immersion of the sample in a clearing medium, minimizing the number of experimental procedures and facilitating the handling of multiple samples.

[0003] Another advantage of the CUBIC reagent is that the amino alcohol can decolorize endogenous heme in whole organ and whole body samples from mice; since its first report, the reagent and protocol have been continuously updated to improve transparency and reduce processing time.

[0004] Traditional immunostaining processes, including slide preparation, mounting, imaging, and data quality, lack assurance (manual slide preparation, data is easily damaged, and registration is difficult), resulting in low axial resolution imaging results.

[0005] Based on the TDI-fMOST three-dimensional optical imaging device, imaging is completed simultaneously with slicing of the whole stained tissue sample. The upper and lower layers of the slice are naturally registered, without human error, and the matching degree is high. Therefore, the accuracy of the reconstructed three-dimensional tissue results is also high, which can better solve the above problems. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a three-dimensional imaging method for biological tissue samples based on CUBIC transparent antibody staining.

[0007] This invention discloses a three-dimensional imaging method for biological tissue samples based on CUBIC clearing antibody staining, comprising:

[0008] Mouse brain tissue was placed in CUBIC-1 solution for defatting and dehydration;

[0009] The defatted and dehydrated mouse brain tissue was subjected to cell membrane permeation treatment.

[0010] The mouse brain tissue, after being permeabilized, was then sealed.

[0011] Use diluted primary antibody GFAP and secondary antibody Alexa Incubate the sealed mouse brain tissue;

[0012] The mouse brain tissue, which had been incubated with antibodies twice, was washed.

[0013] The washed mouse brain tissue was placed in CUBIC-2 solution for matching, and then the mouse brain tissue was embedded in resin.

[0014] The resin-embedded samples were placed in a buffer bath, and images were acquired using a TDI-fMOST camera.

[0015] As a further improvement of the present invention, before defatting and dehydrating the mouse brain tissue, the method further includes:

[0016] Mouse brain tissue was fixed in paraformaldehyde solution, wherein the mass fraction of paraformaldehyde solution was 3-5%, and the fixation time was 2-48 hours.

[0017] As a further improvement of the present invention, the mouse brain tissue is left to stand in CUBIC-1 solution for 1 to 10 days.

[0018] As a further improvement of the present invention, between the defatting and dehydration of mouse brain tissue and the cell membrane permeability treatment, the following is also included:

[0019] Mouse brain tissue was washed with PBS for 1–3 days.

[0020] As a further improvement of the present invention, cell membrane permeabilization treatment of mouse brain tissue was performed using PBS / Triton X-100 / DMSO / glycine solution; wherein the volume fraction of Triton X-100 was 0.001-1%, the volume fraction of DMSO was 2-40%, the molar amount of glycine was 0.001-10M, and the treatment temperature was 30-50℃.

[0021] As a further improvement of the present invention, mouse brain tissue was blocked using a PBS / Triton X / DMSO / goat serum solution; wherein the volume fraction of Triton X-100 was 0.001-1%, the volume fraction of DMSO was 2-40%, the volume fraction of goat serum was 0.001-10%, and the treatment temperature was 30-50°C.

[0022] As a further improvement of the present invention, the primary antibody GFAP was diluted with PBS / Tween-20 / DMSO / goat serum solution; wherein the volume fraction of Tween-20 was 0.001-1%, the volume fraction of DMSO was 2-40%, the volume fraction of goat serum was 0.001-10%, the treatment temperature was 30-50°C, the treatment time was 1-10 days, and the ratio of primary antibody GFAP was 1:(10-10000).

[0023] As a further improvement to the present invention, the secondary antibody Alexa was diluted with PBS / Tween-20 / DMSO / goat serum solution. The volume fraction of Tween-20 was 0.001–1%, the volume fraction of DMSO was 2–40%, the volume fraction of goat serum was 0.001–10%, the treatment temperature was 30–50℃, the treatment time was 1–10 days, and the ratio of secondary antibody GFAP was 1:(10–10000).

[0024] As a further improvement of the present invention, mouse brain tissue that has been incubated with antibodies twice is placed in PBS / Tween-20 solution for washing; wherein the volume fraction of Tween-20 is 0.001-1% and the treatment time is 1-10 days.

[0025] As a further improvement of the present invention, the resin-embedded sample is placed in a 0.05-0.2M PBS buffer bath.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] 1. This invention uses the CUBIC transparency method, which can reduce the number of experimental procedures and process multiple samples at the same time, and can also suppress tissue background fluorescence during dehydration, degreasing and matching.

[0028] 2. This invention utilizes a TDI-fMOST (fluorescence micro-optical tomography) method to simultaneously cut and collect samples from resin-embedded specimens, thereby enabling imaging of the entire CUBIC-transparent antibody-stained mouse brain sample; thus obtaining continuous cross-sectional images of the entire mouse brain at a layer of 1-10 μm. Attached Figure Description

[0029] Figure 1 This is a fine cross-sectional image of a 3mm mouse brain hemisphere provided in Example 1 of the present invention;

[0030] Figure 2 This is a continuous section of the mouse brain hemisphere (3mm) provided in Example 1 of the present invention, with a layer of 1-2μm.

[0031] Figure 3 This is a 3D stereoscopic image of a 3mm mouse brain hemisphere provided in Embodiment 1 of the present invention. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] The present invention will now be described in further detail with reference to the accompanying drawings:

[0034] This invention provides a three-dimensional imaging method for biological tissue samples based on CUBIC clearing antibody staining. This method involves degreasing and dehydrating the biological tissue using CUBIC-1 reagent (composed of urea, ethylenediamine, octylphenol polyoxyethylene ether, and Triton X-100), followed by the use of the primary antibody GFAP, which binds to astrocytes, and the secondary antibody Alexa... Combining GFAP to display green fluorescence, whole-body immunofluorescence staining of biological tissues and organs is performed. Based on the optical matching between CUBIC-2 reagent (CUBIC-2 reagent components are sucrose, urea, 2,2,2'-nitrotriethanol, and Triton X-100) and biological tissues, TDI-fMOST imaging is used to perform continuous section tomographic imaging of whole biological samples. The staining method can clearly show the three-dimensional effect of the internal cellular level of organs, and has the characteristics of being fast, economical, and high-quality.

[0035] Specifically, it includes:

[0036] Step 1: Fix the mouse brain tissue in paraformaldehyde solution; wherein the mass fraction of paraformaldehyde solution is 3-5%, and the fixation time is 2-48 hours; the mouse brain tissue used can be purchased directly as isolated mouse brain tissue, or mouse brain tissue removed after perfusion of PBS into the mouse heart, and the molar amount of the PBS solution is 0.05-0.2M.

[0037] Step 2: Place the mouse brain tissue in CUBIC-1 solution for defatting and dehydration; the mouse brain tissue is left to stand in CUBIC-1 solution for 1 to 10 days.

[0038] Step 3: Wash the mouse brain tissue with PBS; the PBS washing time is 1 to 3 days.

[0039] Step 4: Place the defatted and dehydrated mouse brain tissue in PBS / Triton X-100 / DMSO / glycine solution for cell membrane permeabilization treatment; wherein the volume fraction of Triton X-100 is 0.001-1%, the volume fraction of DMSO is 2-40%, the molar amount of glycine is 0.001-10M, and the treatment temperature is 30-50℃.

[0040] Step 5: Place the cell membrane permeabilized mouse brain tissue in PBS / Triton X / DMSO / goat serum solution for blocking treatment; wherein the volume fraction of Triton X-100 is 0.001-1%, the volume fraction of DMSO is 2-40%, the volume fraction of goat serum is 0.001-10%, and the treatment temperature is 30-50℃.

[0041] Step 6: Incubate the blocked mouse brain tissue with primary antibody GFAP diluted with PBS / Tween-20 / DMSO / goat serum solution; wherein the volume fraction of Tween-20 is 0.001-1%, the volume fraction of DMSO is 2-40%, the volume fraction of goat serum is 0.001-10%, the treatment temperature is 30-50℃, the treatment time is 1-10 days, and the ratio of primary antibody GFAP is 1:(10-10000).

[0042] Step 7: Dilute the secondary antibody Alexa with PBS / Tween-20 / DMSO / goat serum solution. The mouse brain tissue from step 6 was incubated; wherein the volume fraction of Tween-20 was 0.001–1%, the volume fraction of DMSO was 2–40%, the volume fraction of goat serum was 0.001–10%, the treatment temperature was 30–50℃, the treatment time was 1–10 days, and the ratio of secondary antibody GFAP was 1:(10–10000).

[0043] Step 8: Place the mouse brain tissue that has been incubated with antibodies twice in PBS / Tween-20 solution for washing; wherein the volume fraction of Tween-20 is 0.001-1% and the treatment time is 1-10 days.

[0044] Step 9: Place the washed mouse brain tissue in CUBIC-2 solution for matching, and then embed the mouse brain tissue in resin.

[0045] Step 10: Place the resin-embedded sample in a 0.05–0.2 M PBS buffer bath and acquire images using a TDI–fMOST camera (water mirror imaging).

[0046] Example 1:

[0047] A three-dimensional imaging method for biological tissue samples based on CUBIC clearing antibody staining includes:

[0048] S11. Fix mouse brain tissue in 3% paraformaldehyde solution for 24 hours.

[0049] S12. Place the mouse brain tissue in CUBIC-1 and let it stand for 1 day.

[0050] S13. Wash the mouse brain tissue with PBS for 1 day.

[0051] S14. Then, the mouse brain tissue was placed in PBS / Triton X-100 / DMSO / glycine solution for cell membrane permeabilization treatment. The volume fraction of Triton X-100 was 0.001%, the volume fraction of DMSO was 2%, and the molar amount of glycine was 0.001M.

[0052] S15. The mouse brain tissue was placed in PBS / TritonX / DMSO / goat serum solution for blocking treatment. The volume fraction of TritonX-100 was 0.001%, the volume fraction of DMSO was 2%, the volume fraction of goat serum was 0.001%, and the treatment temperature was 30℃.

[0053] S16. Rat brain tissue was incubated with primary antibody (GFAP) diluted with PBS / Tween-20 / DMSO / goat serum solution. The volume fraction of Tween-20 was 0.001%, the volume fraction of DMSO was 2%, the volume fraction of goat serum was 0.001%, the treatment temperature was 30℃, the treatment time was 2 days, and the primary antibody ratio was 1:100.

[0054] S17, Secondary antibody diluted with PBS / Tween-20 / DMSO / goat serum solution (Alexa) Mouse brain tissue was incubated with Tween-20 at a volume fraction of 0.001%, DMSO at a volume fraction of 2%, and goat serum at a volume fraction of 0.001%. The treatment temperature was 30℃, the treatment time was 2 days, and the secondary antibody ratio was 1:100.

[0055] S18. The antibody-incubated mouse brain tissue was washed in PBS / Tween-20 solution with a volume fraction of 0.001% for 2 days.

[0056] S19. The mouse brain tissue was matched by placing it in CUBIC-2 solution, and then the mouse brain tissue was embedded in resin.

[0057] S110. Place the prepared resin-embedded sample in a 0.05M PBS buffer bath and use a TDI-fMOST camera for water mirror imaging to obtain the following results. Figure 3 The image shown is a 3mm three-dimensional image of the mouse brain hemisphere.

[0058] like Figures 1-2 As shown: Mouse brain tissue was sectioned using a vibratory microtome and preliminarily imaged using a microscope; the resin-embedded sample was imaged by simultaneously cutting, rinsing with PBS, and collecting data using a TDI-fMOST (fluorescence microscopic optical tomography) method.

[0059] Example 2:

[0060] A three-dimensional imaging method for biological tissue samples based on CUBIC clearing antibody staining includes:

[0061] S21. Fix mouse brain tissue in 5% paraformaldehyde solution for 48 hours.

[0062] S22. Place the mouse brain tissue in CUBIC-1 for 3 days.

[0063] S23. Wash the mouse brain tissue with PBS for 10 days.

[0064] S24. Then, the mouse brain tissue was placed in PBS / Triton X-100 / DMSO / glycine solution for cell membrane permeabilization treatment. The volume fraction of Triton X-100 was 2%, the volume fraction of DMSO was 10%, and the molar amount of glycine was 1M.

[0065] S25. The mouse brain tissue was placed in PBS / TritonX / DMSO / goat serum solution for blocking treatment. The volume fraction of TritonX-100 was 2%, the volume fraction of DMSO was 10%, the volume fraction of goat serum was 1%, and the treatment temperature was 40℃.

[0066] S26. Rat brain tissue was incubated with primary antibody (GFAP) diluted with PBS / Tween-20 / DMSO / goat serum solution. The volume fraction of Tween-20 was 2%, the volume fraction of DMSO was 10%, the volume fraction of goat serum was 1%, the treatment temperature was 40℃, the treatment time was 5 days, and the primary antibody addition ratio was 1:1000.

[0067] S27. Secondary antibody diluted with PBS / Tween-20 / DMSO / goat serum solution (Alexa) 488) Incubate mouse brain tissue with 2% Tween-20, 10% DMSO, and 1% goat serum at 40°C for 5 days. The secondary antibody ratio is 1:1000.

[0068] S28. The antibody-incubated mouse brain tissue was washed in PBS / Tween-20 solution with a volume fraction of 2% and a treatment time of 3 days.

[0069] S29. Matching was performed using mouse brain tissue placed in CUBIC-2 solution, and then the mouse brain tissue was embedded in resin.

[0070] S210. Place the prepared resin-embedded sample in a 0.05M PBS buffer bath and collect images using a TDI-fMOST camera (water mirror imaging).

[0071] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A three-dimensional imaging method for biological tissue samples based on CUBIC clearing antibody staining, characterized in that, include: Mouse brain tissue was fixed in paraformaldehyde solution; wherein the mass fraction of paraformaldehyde solution was 3-5% and the fixation time was 2-48 hours. Mouse brain tissue was placed in CUBIC-1 solution for defatting and dehydration; the components of CUBIC-1 solution are urea, ethylenediamine, octylphenol polyoxyethylene ether and Triton X-100. The defatted and dehydrated mouse brain tissue was subjected to cell membrane permeabilization treatment using PBS / Triton X-100 / DMSO / glycine solution; wherein the volume fraction of Triton X-100 was 0.001~1%, the volume fraction of DMSO was 2~40%, the molar concentration of glycine was 0.001~10M, and the treatment temperature was 30~50℃. Mouse brain tissue subjected to cell membrane permeabilization was blocked using PBS / Triton X-100 / DMSO / goat serum solution; wherein the volume fraction of Triton X-100 was 0.001~1%, the volume fraction of DMSO was 2~40%, the volume fraction of goat serum was 0.001~10%, and the treatment temperature was 30~50℃. The primary antibody GFAP was diluted with PBS / Tween-20 / DMSO / goat serum solution, and the secondary antibody Alexa Fluor® 488 was diluted with PBS / Tween-20 / DMSO / goat serum solution. The diluted primary antibody GFAP and secondary antibody Alexa Fluor® 488 were used to incubate blocked mouse brain tissue. During the dilution of the primary antibody GFAP, the volume fraction of Tween-20 was 0.001–1%, the volume fraction of DMSO was 2–40%, the volume fraction of goat serum was 0.001–10%, the treatment temperature was 30–50°C, the treatment time was 1–10 days, and the ratio of primary antibody GFAP to secondary antibody was 1:(10–10000). The secondary antibody Alexa Fluor® 488 was diluted with PBS / Tween-20 / DMSO / goat serum solution. During the dilution of Fluor®488, the volume fraction of Tween-20 is 0.001~1%, the volume fraction of DMSO is 2~40%, the volume fraction of goat serum is 0.001~10%, the treatment temperature is 30~50℃, the treatment time is 1~10 days, and the ratio of the secondary antibody Alexa Fluor®488 is 1:(10~10000). The mouse brain tissue, which had been incubated with antibodies twice, was washed. The washed mouse brain tissue was placed in CUBIC-2 solution for optical matching, and then the mouse brain tissue was embedded in resin; the components of CUBIC-2 solution are sucrose, urea, 2,2,2'-nitrotriethanol and Triton X-100. The resin-embedded samples were placed in a buffer bath, and images were acquired using a TDI-fMOST camera.

2. The three-dimensional imaging method for biological tissue samples based on CUBIC clearing antibody staining as described in claim 1, characterized in that, The rat brain tissue was left to stand in CUBIC-1 solution for 1 to 10 days.

3. The three-dimensional imaging method for biological tissue samples based on CUBIC clearing antibody staining as described in claim 1, characterized in that, Between the defatting, dehydration, and cell membrane permeability treatment of mouse brain tissue, the following also applies: Mouse brain tissue was washed with PBS for 1–3 days.

4. The three-dimensional imaging method for biological tissue samples based on CUBIC clearing antibody staining as described in claim 1, characterized in that, Mouse brain tissue that had been incubated with antibodies twice was placed in PBS / Tween-20 solution for washing; the volume fraction of Tween-20 was 0.001-1%, and the treatment time was 1-10 days.

5. The three-dimensional imaging method for biological tissue samples based on CUBIC clearing antibody staining as described in claim 1, characterized in that, Place the resin-embedded samples in a 0.05-0.2M PBS buffer bath.

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