Reagent for reducing autofluorescence and method for preparing animal tissue paraffin flakes

Abstract

The invention discloses a reagent for reducing autofluorescence, which comprises copper sulfate, ammonium chloride and a solvent, and the solvent is water. The invention further discloses a method for preparing the animal tissue paraffin slice. According to the method, cell membrane fluorescence labeling can be maintained while good animal tissue morphology is maintained, non-specific fluorescence is reduced, the signal-to-noise ratio of fluorescence imaging is increased, operation is easy and convenient, reagents are low in cost and easy to obtain, and the imaging quality is effectively improved.

Description

Technical Field

[0001] The invention relates to a reagent for reducing autofluorescence and a method for preparing animal tissue paraffin sections, belonging to the field of biological tissue fluorescence imaging. Background Art

[0002] Adipocyte membrane fluorescence labeling is a commonly used adipocyte identification technology in biomedical research (such as mTmG cell membrane labeling), which is widely used in adipocyte lineage tracing, functional research, metabolic mechanism exploration, and related disease research. Although traditional tissue fixation, dehydration, transparency, wax immersion, and paraffin embedding methods can well preserve the tissue morphology of adipocytes, the fluorescent labeling will be quenched during the tissue preparation process, and the fixation process will also cause significant autofluorescence signals ( Figure 1 A, B, C). In order to avoid the loss of fluorescent labels during tissue preparation, fresh adipose tissue frozen sections are currently used instead of traditional paraffin sections to preserve the fluorescent signal.

[0003] However, there are technical difficulties in the preparation and image acquisition of adipose tissue frozen sections. Adipose tissue is rich in lipids, has a low freezing point, and is not hard enough. It requires high technical skills from the operator of the frozen section machine, and the preservation of tissue morphology is poor, making it difficult to analyze images ( Figure 2 ); In addition, frozen sections of fat are usually thick, and using ordinary fluorescence microscopes to take pictures will result in stacked fat cells, making it difficult to analyze the images ( Figure 3 ), which affects the interpretation of the results and requires further imaging using a laser confocal microscope.

[0004] Autofluorescence is a background fluorescence signal that is unrelated to the target signal and appears when intracellular substances are exposed to the excitation light during fluorescence detection. Adipose tissue (especially brown adipose tissue) is rich in metabolites such as lipofuscin and has significant autofluorescence, which interferes with data interpretation and affects the accuracy and reliability of the results. In addition, during tissue fixation, aldehydes used as fixatives will combine with amines in the tissue to form Schiff bases, introducing exogenous autofluorescence.

[0005] Therefore, a preparation method is needed that can maintain cell membrane fluorescence signals while preserving good tissue morphology, provide technical support for research based on cell membrane fluorescence labeling, and help researchers improve the accuracy and repeatability of experimental results. Summary of the invention

[0006] The purpose of the present invention is to provide a reagent for reducing autofluorescence, which can reduce the nonspecific fluorescence of animal tissue itself, thereby highlighting the fluorescent labeling of cell membranes and enhancing the fluorescent labeling.

[0007] The technical solution adopted by the present invention is:

[0008] A reagent for reducing autofluorescence comprises copper sulfate, ammonium chloride and a solvent, wherein the solvent is water.

[0009] Preferably, the concentration of the copper sulfate is 1.2-2.0 g / L, and the concentration of the ammonium chloride is 2.1-3.0 g / L.

[0010] The use of the above reagents in preparing animal tissue paraffin sections.

[0011] The present invention also discloses a method for preparing animal tissue paraffin sections, which can maintain good tissue morphology while maintaining cell membrane fluorescence signals and reducing tissue nonspecific fluorescence during animal tissue sectioning, and the steps include:

[0012] (1) After dehydration and transparency treatment, the tissue sample is immersed in paraffin for embedding;

[0013] (2) placing the embedded tissue paraffin sections on a glass slide, dewaxing and hydrating the sections, placing the sections in a PBS buffer solution, and then immersing the sections in the reagent for reducing autofluorescence as described in claim 1 or 2, and then washing the sections; the PBS buffer solution can complete the aforementioned hydration effect. In addition, as a buffer solution, PBS also has a certain function of protecting cell membrane fluorescent proteins;

[0014] (3) Remove excess liquid around the adipose tissue slices, add sealing medium to the slices, and cover with a coverslip.

[0015] Preferably, the treatment time with the reagent for reducing autofluorescence in step (2) is 5 to 15 minutes and the temperature is 2 to 8 degrees Celsius.

[0016] Preferably, the dehydration treatment in step (1) refers to treating the adipose tissue in 95% ethanol for 12 to 24 hours, and then dehydrating it in anhydrous ethanol for 3 to 5 times, each time for 0.5 to 2 hours. During this process, the temperature of each solution is 2 to 8 degrees Celsius and protected from light.

[0017] Preferably, the transparent treatment in step (1) refers to immersing the adipose tissue in a xylene solution for 1 to 3 times, each time for 0.5 to 2 hours, at a treatment temperature of 2 to 8 degrees Celsius, and avoiding light.

[0018] Preferably, in step (2), the slice thickness is 4 to 10 microns, and the slice is spread in a water bath at 35 to 45 degrees Celsius and then quickly fished out. The slide containing the tissue slice is placed in an oven for drying at 37 degrees Celsius for 30 minutes in a dark oven.

[0019] Preferably, in step (2), the tissue sections are dewaxed in xylene for 1 to 3 times, each time for 5 minutes, at a temperature of 2 to 8 degrees Celsius, and protected from light;

[0020] The tissue sections were hydrated in 95% ethanol 2 to 3 times for 1 minute each time at a temperature of 2 to 8 degrees Celsius, protected from light;

[0021] Place tissue sections in PBS buffer 2-3 times for 1 min each time at 2-8 degrees Celsius, protected from light.

[0022] Compared with the traditional embedding method, the present invention omits the formaldehyde fixation step in view of the fact that aldehyde fixation will produce significant spontaneous fluorescence; the membrane fluorescent protein will be soluble in low concentration ethanol (such as 75% ethanol), and the present invention also omits the 75% ethanol dehydration step; considering that the membrane fluorescent protein will be quenched at room temperature or light conditions, the present invention completes the ethanol dehydration and xylene transparency process under light-proof conditions of 2 to 8 degrees Celsius.

[0023] Accordingly, the method of the present invention prolongs the dehydration time of high-concentration ethanol (95% ethanol) and the transparent time of xylene to ensure the quality of tissue wax block preparation.

[0024] The present invention provides a reagent and an animal tissue preparation method, which are particularly suitable for the preparation of adipose tissue sections, and can also be used for the preparation of tissue sections of other animal tissues such as skeletal muscle, myocardium, intestine, pancreas, kidney, etc., which can avoid the quenching of cell membrane fluorescence labeling in the traditional paraffin wax preparation process, can retain good tissue morphology, and reduce non-specific autofluorescence signals in the subsequent staining process, thereby improving imaging quality. The reagent and method of the present invention can maintain cell membrane fluorescence labeling while maintaining good tissue morphology, reduce non-specific fluorescence, improve the fluorescence imaging signal-to-noise ratio, and are simple and convenient to operate, and the reagent cost is low and easy to obtain, effectively improving the imaging quality. The method is applicable to adipose tissue and non-adipose tissue in various parts. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Fluorescence images of adipose tissue with cell membrane fluorescent markers after traditional tissue fixation, dehydration, transparency, wax immersion, paraffin embedding, sectioning, dewaxing and sealing. A. Excitation wavelength is 650nm; B. Excitation wavelength is 488nm; C. Excitation wavelength is 358nm. It can be observed that the fluorescent markers on the adipocyte membrane have been quenched. There are significant autofluorescence signals in multiple fluorescence channels.

[0026] Figure 2 This is an image of fresh adipose tissue observed under a fluorescence microscope after freezing. Adipocytes are marked with green membrane fluorescence signals. The slice thickness is 10 microns.

[0027] Figure 3 This is an image of fresh adipose tissue observed under a fluorescence microscope after freezing. Adipocytes are marked with green membrane fluorescence signals. The slice thickness is 40 microns.

[0028] Figure 4 These are images of the fat tissue sections prepared in Example 1 and Comparative Example 1 observed under a fluorescence microscope.

[0029] A. White adipose tissue not treated with an agent that reduces autofluorescence. B. Brown adipose tissue not treated with an agent that reduces autofluorescence. C. White adipose tissue treated with an agent that reduces autofluorescence. D. Brown adipose tissue treated with an agent that reduces autofluorescence.

[0030] Figure 5 These are images of pancreatic tissue sections observed under a fluorescence microscope.

[0031] Figure 6 These are images of heart tissue sections observed under a fluorescence microscope.

[0032] Figure 7 These are images of skeletal muscle tissue sections observed under a fluorescence microscope.

[0033] Figure 8 These are images of kidney tissue sections observed under a fluorescence microscope.

[0034] Fig. 9 These are images of digestive tract tissue sections observed under a fluorescence microscope.

[0035] Fig.10 This is an image of a vascular tissue section observed under a fluorescence microscope. DETAILED DESCRIPTION

[0036] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation mode, structure, characteristics and effects of the present invention are described in detail below in combination with the accompanying drawings and preferred embodiments.

[0037] Example 1

[0038] The method for preparing paraffin sections of adipose tissue with retained membrane fluorescence labeling comprises the following steps:

[0039] Step 1: Obtain adipose tissue sample.

[0040] Step 1.1: After anesthesia, the mouse (C57BL / 6J) was opened, the right atrial appendage was cut open, and PBS buffer was perfused into the left ventricle;

[0041] Step 1.2, separate fat from various parts and select a white fat tissue and a brown fat tissue;

[0042] Step 2, preparing tissue wax blocks: the samples obtained in the above step 1 are dehydrated, transparentized, wax-impregnated and embedded to finally obtain tissue wax blocks.

[0043] Step 2.1: After the separated fat is placed in 95% ethanol overnight (12-18 hours), it is placed in anhydrous ethanol for dehydration 4 times (1 hour each time). During this process, the temperature of each solution is 4 degrees Celsius and protected from light.

[0044] Step 2.2: Transparent the adipose tissue treated in step 2.1 in xylene solution twice (1 hour each time) at 4 degrees Celsius, protected from light.

[0045] Step 2.3: Immerse the adipose tissue treated in step 2.2 in paraffin wax for 4 times (1 hour each time). The temperature of the paraffin solution is 65 degrees Celsius and protected from light.

[0046] Step 3: Cut the tissue wax block obtained in step 2 into 5-micron paraffin sections.

[0047] Step 4: Place the paraffin sections obtained in step 3 into a reagent that reduces autofluorescence to dewax and remove non-specific fluorescence.

[0048] Step 4.1: Use a rotary paraffin slicer to slice the adipose tissue into paraffin slices with a thickness of 5 microns. Spread the slices in a 40-degree Celsius water bath and quickly remove the slices. Dry the slides with the tissue slices in an oven for 30 minutes at 37 degrees Celsius, away from light.

[0049] Step 4.2: Dewax the paraffin sections in step 4.1 in xylene twice (5 minutes each time) at 4 degrees Celsius in the dark.

[0050] Step 4.3: Place the paraffin sections in 4.2 in 95% ethanol three times (1 minute each time) at 4 degrees Celsius and protect from light.

[0051] Step 4.4: Place the paraffin sections in 4.3 in PBS buffer three times (1 minute each time) at 4 degrees Celsius and protect from light.

[0052] Step 4.5: Place the paraffin sections in step 4.4 in a reagent that reduces autofluorescence for 10 minutes at 4 degrees Celsius, and then place in deionized water for later use. During the process, the temperature of each solution is 4 degrees Celsius and protected from light.

[0053] Step 5: Seal the slides with a sealing medium and take images.

[0054] Step 5.1: Remove excess liquid around the sample, add sealing medium on the tissue section, and cover with a coverslip.

[0055] Step 5.2: Air-dry the sealed tissue sections for 5 minutes and image them using a fluorescence microscope. The imaging results are as follows: Figure 4 C and Figure 4 As shown in D.

[0056] Figure 1 The conventional paraffin sectioning process was used, and it can be observed that the fluorescent markers on the adipocyte membrane have been quenched. There are significant autofluorescence signals in multiple fluorescence channels. Figure 2 The frozen sections of adipose tissue used (thin sections, 10 micrometers thick) showed that the morphology of adipocytes was poorly preserved, making image analysis difficult. Figure 3 The frozen sections of adipose tissue used (thick sections, 40 microns thick) allow observation of stacked adipocytes, which are difficult to analyze in images. Figure 1 to Figure 4 It can be seen that the method of the present invention can avoid the quenching of cell membrane fluorescence labeling in the traditional paraffin sectioning process, can retain good adipose tissue morphology, and can reduce nonspecific autofluorescence signals in the subsequent staining process, thereby improving imaging quality.

[0057] Comparative Example 1

[0058] The method of this comparative example is basically the same as that of Example 1, except that the treatment in step 4.5 is not performed. The imaging results of the obtained slices under a fluorescence microscope are as follows: Figure 4 A and Figure 4 As shown in B.

[0059] Example 2

[0060] The same method as in Example 1 was used to prepare slices of pancreas, heart, skeletal muscle, kidney, digestive tract tissue, and vascular tissue of mice, and fluorescence microscopy was used for imaging. The imaging results are shown in FIG. Figures 5 to 10 As shown, the mice are gene-edited mice with cell membrane fluorescence signals. It can be seen that the cell morphology is well preserved and the cell membrane fluorescence signals are clearly visible.

[0061] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A reagent for reducing autofluorescence, characterized in that The invention comprises copper sulfate, ammonium chloride and a solvent, and the solvent is water.

2. The reagent according to claim 1, characterized in that The concentration of the copper sulfate is 1.2-2.0 g / L, and the concentration of the ammonium chloride is 2.1-3.0 g / L.

3. Use of the reagent described in claim 1 or 2 in preparing animal tissue paraffin sections.

4. A method for preparing animal tissue paraffin sections, characterized in that: The steps include: (1) After dehydration and transparency treatment, the animal tissue samples are immersed in paraffin for embedding; (2) placing the embedded tissue paraffin sections on a glass slide, dewaxing and hydrating them, repairing them in PBS buffer, and then immersing them in the reagent for reducing autofluorescence as described in claim 1 or 2, and then washing the tissue sections; (3) Remove excess liquid around the tissue section, add sealing medium on the section, and cover with a coverslip.

5. The method according to claim 4, characterized in that The time for treating with the reagent for reducing autofluorescence in step (2) is 5 to 15 minutes, and the temperature is 2 to 8 degrees Celsius.

6. The method according to claim 4, characterized in that The dehydration treatment in step (1) refers to treating the tissue in 95% ethanol for 12 to 24 hours and then dehydrating it in anhydrous ethanol for 3 to 5 times, each time for 0.5 to 2 hours. During this process, the temperature of each solution is 2 to 8 degrees Celsius and protected from light.

7. The method according to claim 4, characterized in that The transparent treatment in step (1) refers to immersing the tissue in a xylene solution for 1 to 3 times, each time for 0.5 to 2 hours, at a treatment temperature of 2 to 8 degrees Celsius, and avoiding light.

8. The method according to claim 4, characterized in that In step (2), the slice thickness is 4 to 10 microns. After spreading the slice in a water bath at 35 to 45 degrees Celsius, the slice is quickly fished out and the slide containing the tissue slice is placed in an oven for drying at 37 degrees Celsius for 30 minutes in a dark oven.

9. The method according to claim 4, characterized in that In step (2), the tissue sections are dewaxed in xylene for 1 to 3 times, each time for 1 to 10 minutes, at a temperature of 2 to 8 degrees Celsius, and protected from light; The tissue sections were hydrated in 95% ethanol 2 to 3 times for 1 minute each time at a temperature of 2 to 8 degrees Celsius, protected from light; Place tissue sections in PBS buffer 2-3 times for 1 min each time at 2-8 degrees Celsius, protected from light.

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