Quantitative analysis method for axon-somatic synapse cell membrane protein

Abstract

The invention relates to a quantitative analysis method for axon-somatic synapse cell membrane protein. The method comprises the following steps: performing layer scanning on neuronal cells subjected to immunofluorescence staining to obtain a confocal picture; selecting an image of a region of interest on a cell membrane through Fiji software, and straightening the image to obtain a linear cell membrane protein distribution image of RGB three channels; converting the obtained original image into binary images, overlapping the two binary images together to form a synthetic image, and displaying distribution and co-localization information of different membrane proteins on the synthetic image; membrane proteins on the synthesized image are counted and marked in size through Fiji software. According to the method, co-localization, counting and size information on the synaptic cell membrane protein can be quickly obtained on the original fluorescent staining picture of the neuronal cell by utilizing Fiji software. Therefore, research and analysis of related proteins can be more efficient, more convenient and more accurate.

Description

Technical Field

[0001] The present invention relates to the field of neuroscience and is used for analyzing the detection, counting and size measurement of membrane proteins on neuronal cell membranes after immunofluorescence staining, as well as the co-localization analysis of two fluorescent signals. The present invention is mainly suitable for the co-localization and quantitative analysis of axon-cell body synapses. Background Art

[0002] Neural signal transmission is one of the most important basic biological processes in living organisms, which helps promote proper coordination and interaction between various parts of the body. The role of cell membrane proteins in neural signal transmission has become one of the most concerned issues in the field. In the case of neural signal transmission, the role of cell membrane proteins is not limited to maintaining the integrity and stability of the cell membrane, but also mainly controls the secretion and rapid transmission of neurotransmitters. Therefore, the study of neural signal transmission is inseparable from the study of neuronal cell membrane proteins.

[0003] Currently, the separation of neuronal cell membrane proteins is still in the field of proteomics, which requires the use of proteomics methods to extract and purify target proteins for subsequent research. The process is complex and lengthy, and the interactions, co-localization, and distribution of multiple membrane proteins cannot be known.

[0004] Colocalization analysis of presynaptic and postsynaptic membrane proteins is an important technique in neuroscience research, mainly used to understand the signal transmission mechanism between neurons. It helps to understand the interactions between different membrane proteins and their roles in synaptic function. By analyzing the colocalization changes of membrane proteins, it can help understand the pathogenesis of neurodegenerative diseases, mental illnesses, etc., and identifying the interactions of key membrane proteins can also provide a basis for the targets of new drugs. Summary of the invention

[0005] The purpose of the present application is to provide a quantitative analysis method for axon-soma synaptic cell membrane proteins, aiming to solve the problems existing in the prior art.

[0006] The present application embodiment provides a method for quantitative analysis of axon-cell body synaptic cell membrane proteins, comprising the following steps:

[0007] S1. Perform immunofluorescence staining on neuronal cell slices and obtain RGB three-channel confocal images by layer scanning;

[0008] S2. Select a 2D image with an axon-cell body synaptic structure, manually select an area of ​​interest on the cell membrane using Fiji software, and straighten the selected image to obtain a linear cell membrane protein distribution image under RGB three-color channels;

[0009] S3, based on step S2, converting the obtained original image into a binary image, and then overlapping the two binary images to form a composite image, which displays the distribution and co-localization information of different membrane proteins;

[0010] S4. Quantitative analysis of membrane proteins on the composite images was performed using Fiji software, and the generated data results included the following parameters: the area and perimeter of the image of the selected region of interest, the measured values ​​of the number and size of presynaptic and postsynaptic proteins, and the size of the colocalization area.

[0011] Furthermore, the membrane proteins include presynaptic membrane proteins VIAAT and GlyT2 and postsynaptic membrane proteins Gephyrin, GABAARs and GlyR clusters.

[0012] Furthermore, when selecting the region of interest image, immunostaining positive fluorescent spots of both presynaptic and postsynaptic membranes should be selected, and non-specific immunofluorescence spots should be avoided.

[0013] Furthermore, when detecting the size of membrane proteins, a lower limit value of the membrane protein size is set to exclude false positive results.

[0014] Furthermore, when colocalizing, the algorithm for the size of the colocalized fluorescent spot is size>30%·kμm 2 , where k is the lower limit of membrane protein size, and 30% is defined as mature synapses, that is, there is at least 30% overlap between the fluorescent spots on the presynaptic and postsynaptic membranes.

[0015] Furthermore, in step S3, the original image is converted into a binary image using the binary quantization function of the software, and the 16-bit confocal image is converted into an 8-bit image, and the grayscale image is converted into a black-and-white image, wherein all-black or all-white dots can represent the expression and non-expression of the protein.

[0016] The beneficial effect of the present invention is that the present invention can use Fiji software to quickly obtain the co-localization, counting and size information on synaptic cell membrane proteins on the original fluorescent staining images of neuronal cells, thereby making the research and analysis of related proteins more efficient, convenient and accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a confocal image of neurons after fluorescent immunostaining in one embodiment (including selected linear area images).

[0018] Figure 2 The RGB mode image is obtained by straightening the selected linear region image.

[0019] Figure 3 It is the composite image after binary images are merged.

[0020] Figure 4 A composite image with count marks.

[0021] Figure 5 This is the result data graph output by the software.

[0022] Figure 6 This is a diagram showing the effects of membrane protein detection, size measurement and co-localization analysis in another embodiment. DETAILED DESCRIPTION

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] A quantitative analysis method for axon-cell body synaptic cell membrane proteins comprises the following steps:

[0025] S1. Immunofluorescence staining was performed on the sections of neuronal cells, and confocal images of RGB three channels were obtained by layer scanning. The neuronal cell membrane proteins appeared as fluorescent spots in the images after staining.

[0026] S2. Import all confocal images into Fiji software, select 2D images with axon-cell body synaptic structures, and manually select the linear area images of interest on the cell membrane using Fiji software, such as Figure 1 The yellow line shown has a line width of 20 pixels. When selecting the line area of ​​interest, you should choose immunostained positive fluorescent spots with both presynaptic and postsynaptic membranes, and avoid nonspecific immunofluorescent spots.

[0027] The selected image is straightened by Fiji to obtain a linear cell membrane protein distribution image under the RGB three-color channel, such as Figure 2 As shown, displayed in RGB color mode, the red fluorescent dots under the red channel represent GlyR (glycine receptor), and the blue fluorescent dots under the blue channel represent GlyT2 (a transporter on the presynaptic membrane, which is responsible for reabsorbing glycine from the synaptic cleft back into the presynaptic neuron).

[0028] S3, based on step S2, convert the obtained original image into a binary image; the original image is converted into a binary image using the binary quantization function (8bit, 0-255) of the Fiji software, converting the 16-bit confocal image into an 8-bit image, converting the grayscale image into a black-and-white image, in which the value of each fluorescent point is either completely black or completely white. In gene expression data analysis, binary quantification can classify gene expression levels into two categories: "expressed" (1) and "unexpressed" (0).

[0029] The two binary images are then overlapped to form a composite image, which displays the distribution and co-localization information of different membrane proteins. Figure 3 As shown, the red mark represents GlyR, the green mark represents GlyT2, and the yellow mark represents the co-localization site of GlyR-GlyT2.

[0030] S4. Quantitative analysis of membrane proteins on the composite images was performed using Fiji software. Figure 4 Generate data results; the data results include the area and perimeter of the selected region of interest image, the number and size of presynaptic and postsynaptic proteins, and the size of the colocalization area, as shown in Figure 2. Figure 5 shown.

[0031] The membrane proteins applicable to the present invention include presynaptic membrane proteins VIAAT and GlyT2 and postsynaptic membrane proteins Gephyrin and GABA A Rs and GlyR clusters.

[0032] When detecting the size of membrane proteins, the lower limit of the membrane protein size was set according to the references to exclude false positive results. For example, the lower limit of the area of ​​GlyT2 clusters and GlyRs clusters was 0.2 μm 2 and 0.02 μm 2 .

[0033] When co-localized, the size of the co-localized fluorescent spot is calculated as size>30%·kμm 2 , where k is the lower limit of the membrane protein size. For example, when calculating the colocalization of GlyRs clusters, the algorithm formula is size>30%·0.02μm 2 =0.006μm 2 30% was defined as a mature synapse, i.e., there was at least 30% overlap of fluorescent spots on the presynaptic and postsynaptic membranes.

[0034] According to the above process, the motor neurons are analyzed and tested, such as Figure 6As shown, in the figure, A1 represents motor neurons (blue channel), A2 represents presynaptic membrane protein VIAAT (green channel), A3 represents postsynaptic membrane protein Gephyrin (red channel), and A4 represents a synthetic image. The band image (dashed box) in A1-A4 is a selected linear area image, which represents the detection, co-localization, and quantitative analysis of presynaptic and postsynaptic membrane proteins in a section of the cell body and proximal dendrites on the motor neuron. In the figure, A1.1, A2.1, A3.1 and A4.1 all represent original images, and A1.2, A2.2, A3.2 and A4.2 represent the effect display diagram of membrane protein detection after running Fij i. By comparing the original image and the effect diagram after detection, it is found that this method can objectively and accurately detect the target protein.

[0035] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and range of equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A quantitative analysis method for axon-cell body synaptic cell membrane proteins, It is characterized in that The following steps are involved: S1. Perform immunofluorescence staining on neuronal cell slices and obtain RGB three-channel confocal images by layer scanning; S2. Select a 2D image with an axon-cell body synaptic structure, manually select an area of ​​interest on the cell membrane using Fiji software, and straighten the selected image to obtain a linear cell membrane protein distribution image under RGB three-color channels; S3, based on step S2, converting the obtained original image into a binary image, and then overlapping the two binary images to form a composite image, which displays the distribution and co-localization information of different membrane proteins; S4. Quantitative analysis of membrane proteins on the composite images was performed using Fiji software, and the generated data results included the following parameters: the area and perimeter of the image of the selected region of interest, the measured values ​​of the number and size of presynaptic and postsynaptic proteins, and the size of the colocalization area.

2. The quantitative analysis method for axon-soma synaptic cell membrane proteins according to claim 1, It is characterized in that Applicable to inhibitory synapses, including presynaptic membrane proteins VIAAT, GlyT2 and postsynaptic membrane proteins Gephyrin, GABA A Rs and GlyR clusters.

3. The quantitative analysis method for axon-soma synaptic cell membrane proteins according to claim 1, It is characterized in that When selecting the region of interest image, immunostaining positive fluorescent spots with both presynaptic and postsynaptic membranes should be selected, and non-specific immunofluorescence spots should be avoided.

4. The quantitative analysis method for axon-soma synaptic cell membrane proteins according to claim 1, It is characterized in that When detecting the size of membrane proteins, a lower limit value of the membrane protein size was set to exclude false positive results.

5. The quantitative analysis method for axon-soma synaptic cell membrane proteins according to claim 1, It is characterized in that In case of co-localization, the algorithm for the size of co-localized fluorescent spots is size>30%·kμm 2 , where k is the lower limit of membrane protein size, and 30% is defined as mature synapses, that is, there is at least 30% overlap between the fluorescent spots on the presynaptic and postsynaptic membranes.

6. The quantitative analysis method for axon-soma synaptic cell membrane proteins according to claim 1, It is characterized in that In step S3, the original image is converted into a binary image. The binary quantization function of the software is used to convert the 16-bit confocal image into an 8-bit image, and the grayscale image is converted into a black-and-white image, in which all-black or all-white points can represent the expression and non-expression of the protein.

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