CN113466014A - Method for detecting neuron dendritic spine morphology in brain slice - Google Patents

Method for detecting neuron dendritic spine morphology in brain slice Download PDF

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CN113466014A
CN113466014A CN202110750048.4A CN202110750048A CN113466014A CN 113466014 A CN113466014 A CN 113466014A CN 202110750048 A CN202110750048 A CN 202110750048A CN 113466014 A CN113466014 A CN 113466014A
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brain
dil
phosphate buffer
dendritic spines
buffer solution
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吴佳慧
陈婵
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West China Hospital of Sichuan University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34—Purifying; Cleaning
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G01N2001/2873—Cutting or cleaving
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N2001/302—Stain compositions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N2001/305—Fixative compositions

Abstract

The invention belongs to the field of brain neuron research, and particularly relates to a method for detecting the form of neuron dendritic spines in brain slices. The invention provides a rapid detection method for the form of neuron dendritic spines in brain slices, which aims to solve the technical problems of complex steps, long process, high cost, need of using highly toxic reagents and special instruments which are harmful to human bodies in the existing method for staining the dendritic spines in the brain. According to the invention, the low-concentration dye and the high-intensity vortex are adopted, so that the DIL working solution forms extremely tiny particles, and high background interference caused by DIL solid dye residue is avoided by controlling the contact time of the DIL working solution and the brain slice and a proper observation time window, thereby clearly distinguishing single neuron cells. The method greatly simplifies the original DIL scattering method flow, does not need special instruments, has simple operation steps and stable dyeing effect, and is suitable for being popularized in laboratories.

Description

Method for detecting neuron dendritic spine morphology in brain slice
Technical Field
The invention belongs to the field of brain neuron research, and particularly relates to a method for detecting the form of neuron dendritic spines in brain slices.
Background
Dendritic spine (Dendrite spine) is located in the synapse-forming site of neuron dendrites and is closely related to learning and memory. Dendritic spines are critical in the connection between neurons in the brain. As the disease progresses, dendritic spines produce changes in both quantity and morphology, which have a significant impact on brain function. Explaining the altered plasticity of the dendritic spines under pathological conditions helps us to understand the learning and memory processes of the normal brain and the pathogenesis of various nerve-related diseases.
Visualization of the morphology of the dendritic spines of nerve cells for subsequent analysis of the morphology and density of the dendritic spines is currently an internationally recognized important indicator for assessing synaptic connectivity and synaptic plasticity changes. Therefore, staining of the neuronal dendritic spines is widely present in neuroscience research.
The earliest and most classical method of neuronal staining of brain sections was the Golgi staining method (Golgi staining). The main steps of the Golgi dyeing method are as follows: preparing a Golgi solution (potassium dichromate, mercury bichromate and potassium chromate) 5 days in advance, perfusing normal saline after the animal is anesthetized until blood is eliminated, taking the brain, adding the Golgi solution into the brain tissue, and standing for 14 days in a dark place. Then the brain tissue is transferred into 30% sucrose solution until the brain tissue sinks to the bottom, and then the brain tissue is cut into 200-300 mu m brain slices by a vibratory microtome. Brain pieces were transferred to gelatin-coated glass slides and stained by the following procedure: distilled water for 1min, ammonium hydroxide solution for 30min, distilled water for 1 dish, kodak stationary liquid for 30min, distilled water for 1min, gradient dehydration with 50% -100% ethanol for 6 times, placing in CXA solution for 15min, and observing with neutral resin sealing sheet. At present, the Golgi staining kit is sold in the market, but the kit only simplifies the preparation of partial reagents, the steps of preparing solution, soaking for 14 days, dehydrating and staining are still needed, and the reagents are still highly toxic and need to be prepared in a fume hood. The Golgi dyeing method has long experimental process, and the average time is about 2-3 weeks according to different dyeing schemes; the reagent is extremely toxic, and a large amount of heavy metal salts such as mercuric chloride, potassium dichromate and the like and strong carcinogens are needed in the dyeing process.
With the use and development of fluorescence microscopy, methods of staining cells with fluorescent DIL dye and detecting the morphology of dendritic spines under fluorescence microscopy appeared. DIL (1,1 ' -Dictatacyl-3, 3,3 ', 3 ' -tetramethylenecarbacyanine perchlorate, C59H97ClN2O4) Is a lipophilic fluorescent dye. DIL dye is fixed by living cells or fixed cellsThe cell membrane of the cell is taken up into the cell and spreads within the cell over time until the entire cell is filled. At this time, the intact morphology of the whole nerve cells was observed under a fluorescence microscope, and the minute structures such as the spiny tree were recognized. DIL dyes are commonly used as cellular membrane fluorescent molecular probes for staining of cultured cells in vitro. However, the form of the neuron cells cultured in vitro is very different from that of the brain, and the pathological changes of the brain cannot be accurately reflected. Pathological analysis of dendritic spines still needs to be performed in brain sections. Namely, the DIL scattering method requires special equipment such as a gene gun warhead preparation workbench and a gene gun, and is complex to operate, expensive in price and difficult to widely develop.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the existing method for dyeing the dendritic spines in the brain has the problems of complex steps, long process, high cost, and the need of using highly toxic reagents and special instruments which are harmful to human bodies.
The technical scheme adopted by the invention for solving the technical problems is as follows: provides a method for detecting the form of neuron dendritic spines in brain slices, which comprises the following steps:
A. preparing brain tablets: pouring a paraformaldehyde-phosphate buffer solution with the mass volume ratio of 1.5-2% into an experimental animal for 10-20 min, taking the brain and slicing;
B. preparing a DIL working solution: dissolving and diluting a DIL solid into a stock solution with the concentration of 15-25 mmol/L by using an organic solvent, and then diluting the stock solution into a DIL working solution with the concentration of 2-5 mu mol/L by using a phosphate buffer solution; swirling DIL working solution for more than 5 min;
dil staining: flatly paving the brain slices rinsed by the phosphate buffer solution in the DIL working solution, and standing and incubating for 30-60 min in a dark place;
D. and (4) sealing sheet observation: rinsing the DIL-dyed brain slices with a phosphate buffer solution, and sealing; taking 6-12 h as an observation time window.
Further, the concentration of the phosphate buffer solution in the step A is 0.01mol/L, and the pH range is 7.2-7.4; preferably the pH is 7.4.
Further, the specific operations of the perfusion in the step A are as follows: after the experimental animal is deeply anesthetized, perfusing the heart with a phosphate buffer solution for more than 5min, and perfusing the heart with a 1.5-2% paraformaldehyde-phosphate buffer solution for 10-20 min; preferably, the perfusion time is 20 min.
Further, the thickness of the slice in the step A is 100-300 μm; preferably 150 μm thick.
Further, the vortex in the step B is to disperse the DIL working solution by a vortex oscillator; the rotation speed of the vortex oscillator is 1200-2000 rpm/min.
Further, the rinsing operation in step C is: removing the phosphate buffer solution, adding fresh phosphate buffer solution, soaking for more than 5min, and repeating for more than 3 times.
Further, the standing incubation time in the step C is 30-45 min.
Further, the rinsing operation in step D is: washing brain slice with phosphate buffer solution for more than 3 times, each time for more than 5min, and washing away from light.
The invention has the beneficial effects that: according to the invention, the low-concentration dye and the high-intensity vortex are adopted, so that the DIL working solution forms extremely tiny particles, and high background interference caused by DIL solid dye residue is avoided by controlling the contact time of the DIL working solution and the brain slice and a proper observation time window, thereby clearly distinguishing single neuron cells. The method greatly simplifies the original DIL scattering method process, only needs a vibrating microtome and a vortex oscillator, does not need other special instruments, has simple operation steps and stable dyeing effect, and is suitable for being popularized in laboratories.
Drawings
FIG. 1 shows neurons from brain sections stained by DIL scattering, showing high background fluorescence from DIL solid dye.
FIG. 2 is a photograph of hippocampal brain neurons stained using the method of the present invention, with granular images of DAPI-stained nuclei.
Detailed Description
The principle of DIL liquid dye staining is that the dye comes into direct contact with the cell membrane, enters the cell membrane and diffuses throughout the cell along the cell membrane. If the observation of the dendritic spines is to be successful, it is required that both the DIL liquid dye has sufficient time to diffuse throughout the neurons and that only a small number of neurons are stained. However, DIL dye is a non-selective dye, which results in any cell membrane being absorbed, and therefore, if the concentration of the dye is too high, or the staining time is too long, the DIL dye will contact too much brain slices, staining a large number of cells, and causing strong background fluorescence.
Therefore, the invention adopts the low-concentration dye, the dye is swirled to form tiny particles, the dye particles are in temporary contact with brain slices, only a small amount of DIL dye is absorbed by cell membranes, most of redundant liquid DIL dye can be washed away without remaining in the brain slices, and then observation is carried out in a reasonable time window, so that single neuron cells can be clearly distinguished.
The invention provides a rapid detection method of the form of neuron dendritic spines in brain slices, which comprises the following specific steps:
A. preparing brain tablets: injecting a paraformaldehyde-PBS (phosphate buffer solution) with the mass volume ratio of 1.5-2% into the rodent in a light fixation mode for 10-20 min, taking the brain and slicing; soaking the brain slices in PBS solution for later use;
B. preparing a DIL working solution: fully diluting a DIL solid into a stock solution with the concentration of 15-25 mmol/L by using DMSO (dimethyl sulfoxide), and then diluting the stock solution into a DIL working solution with the concentration of 2-5 mu mol/L by using a PBS solution; swirling DIL working solution for more than 5 min;
dil staining: flatly spreading the brain slices rinsed by the PBS solution in the DIL working solution, and standing and incubating for 30-60 min at room temperature in a dark place; since the brain piece is light and thin like a membrane, the brain piece is not folded when transferred with a small brush pen, and the folded part cannot be dyed successfully.
D. And (4) sealing sheet observation: rinsing the DIL-stained brain slices with a PBS solution, and sealing; because the DIL dye can continuously diffuse in the brain slice, if the time is too long, the background fluorescence becomes higher and higher, and 6-12 h is taken as the time window for observation and photographing.
Further, the PBS solution in the step A is 0.01mol/L phosphate buffer solution, and the pH range is 7.2-7.4, preferably 7.4.
Further, the specific operations of the perfusion in the step A are as follows: after the rodent is deeply anesthetized, the rodent is perfused for more than 5min through the heart by using a PBS solution, and then perfused and fixed for 10-20 min, preferably 20min by using a 1.5-2% paraformaldehyde-PBS solution.
Further, the thickness of the slice in the step A is 100-300 μm, and preferably 150 μm. Less than 100 μm complete neurons are too few, and more than 300 μm slices are too thick, so that multi-layer neuron overlap easily occurs, and subsequent image acquisition is influenced.
Further, the vortex in the step B is to disperse the DIL working solution by a vortex oscillator; the rotation speed of the vortex oscillator is 1200-2000 rpm/min. The DIL working solution is required to be prepared before each dyeing, and the working solution is immediately used for dyeing after being swirled and cannot be placed for a long time. DIL precipitation can appear in the centrifuging tube pipe wall of configuration working solution, can not reuse.
Further, the rinsing operation in step C is: sucking out PBS soaked brain slice, adding fresh PBS solution, soaking for more than 5min, and repeating for more than 3 times.
Further, the time of the standing incubation in the step C is preferably 30min to 45 min. There is a high success rate in this range.
Further, the rinsing operation in step D is: washing brain slice with PBS for more than 3 times, each time for more than 5min, and washing away from light.
The dye has not been fully diffused to the whole neuron observed before 6h, and even if the brain slice is washed and mounted, the residual DIL dye on the brain slice can be continuously diffused after 12h, so that the stained cells are increased, and the dendritic spines are overlapped with each other and difficult to analyze. Therefore, the optimal observation time window of the method is 6-12 h.
Example 1
1. Preparation of brain tablet
1.1 preparation of 1.5% strength paraformaldehyde-PBS solution: 15g of paraformaldehyde was dissolved in 1L of a PBS solution (0.01mol/L of phosphate buffer, pH 7.4, hereinafter referred to as PBS all the same solution).
1.2 mice were deeply anesthetized, perfused with PBS for 5min via the heart, then fixed with 1.5% paraformaldehyde-PBS for 20 min.
1.3 the whole brain was removed from the cranial cavity and immediately sectioned on a vibrating microtome at a thickness of 300 μm. After slicing, 1 clean six-well plate was taken and brain slices were soaked in PBS solution for use.
DIL dyeing
2.1 preparation of DIL stock solution: DIL solid was diluted well with DMSO to a 20mmol/L stock, which was frozen at-20 ℃.
2.2 preparing DIL working solution: dil stock of 20mmol/L was diluted to 5. mu.M DIL working solution in a 1.5ml EP tube with PBS solution, and 500. mu.L of working solution was used for each brain piece to completely cover the brain piece.
2.3 vortex: the prepared 1.5ml EP tube containing the working solution is placed on a vortex oscillator to be sufficiently oscillated for 5min, so that the DIL dye in the EP tube forms particles as small as possible. The DIL working solution is required to be prepared before each dyeing, and the working solution is immediately used for dyeing after being swirled and cannot be placed for a long time. The wall of the EP pipe provided with the working fluid can generate DIL precipitation and cannot be used again.
2.4 rinsing brain slices: old PBS soaked in brain slices in a six-hole plate is carefully sucked out by using a pipette gun, and is soaked for 5min after a fresh PBS solution is added, and the process is repeated for 3 times. To remove residual paraformaldehyde.
2.5 prepare a new 24-well cell culture plate. Each well of the 24-well plate was charged with 500ul of the already vortexed DIL working solution.
2.6 carefully transfer the brain slices to be stained from the PBS solution into a 24-well plate with a brush pen and spread flat.
2.7 standing at room temperature in dark place for 1 h.
2.8 washing the brain slices with PBS solution for 3 times, 10min each time, placing the 24-well plate on a shaker during washing, and taking care to avoid light.
2.9 sealing piece: mounting was performed with Mounting Medium with DAPI, Fluoroshield (ab 104139).
3. Observe and take a picture
3.1 sections were stored in the dark at room temperature, and single neuronal cells were observed within 6-12 hours after completion of mounting due to continuous diffusion of DIL dye in the cells.
3.2 photograph: a laser confocal microscope was used and whole cells were photographed in Z-axis mode. The results are shown in FIG. 2.
As can be seen from the figure, the brain slice is dyed by the method of the invention and observed under a proper time window, so that high background interference caused by residual DIL solid dye can be avoided, and single neuron cells can be clearly distinguished.

Claims (8)

1. The method for detecting the morphology of the dendritic spines of the neurons in the brain slices is characterized by comprising the following steps of:
A. preparing brain tablets: pouring a paraformaldehyde-phosphate buffer solution with the mass volume ratio of 1.5-2% into an experimental animal for 10-20 min, taking the brain and slicing;
B. preparing a DIL working solution: dissolving and diluting a DIL solid into a stock solution with the concentration of 15-25 mmol/L by using an organic solvent, and then diluting the stock solution into a DIL working solution with the concentration of 2-5 mu mol/L by using a phosphate buffer solution; swirling DIL working solution for more than 5 min;
dil staining: flatly paving the brain slices rinsed by the phosphate buffer solution in the DIL working solution, and standing and incubating for 30-60 min in a dark place;
D. and (4) sealing sheet observation: rinsing the DIL-dyed brain slices with a phosphate buffer solution, and sealing; taking 6-12 h as an observation time window.
2. The method for detecting the morphology of the dendritic spines in the neurons in the brain slices according to claim 1, wherein the concentration of the phosphate buffer solution in the step A is 0.01mol/L, and the pH range is 7.2-7.4; preferably the pH is 7.4.
3. The method for detecting the morphology of the dendritic spines of the neurons in the brain slice according to claim 1 or 2, wherein the perfusion in the step A is specifically performed by the following steps: after the experimental animal is deeply anesthetized, perfusing the heart with a phosphate buffer solution for more than 5min, and perfusing the heart with a 1.5-2% paraformaldehyde-phosphate buffer solution for 10-20 min; preferably, the perfusion time is 20 min.
4. The method for detecting the morphology of neuronal dendritic spines in a section of a brain according to any one of claims 1 to 3, wherein the thickness of the section in step A is 100 to 300 μm; preferably, the slice thickness is 150. mu.m.
5. The method for detecting the morphology of the dendritic spines in the neurons of the brain slice according to any one of claims 1 to 4, wherein the vortexing in the step B is performed by dispersing DIL working solution by a vortexer; the rotation speed of the vortex oscillator is 1200-2000 rpm/min.
6. The method for detecting the morphology of the dendritic spines of the neurons in the brain slice according to any one of claims 1 to 5, wherein the rinsing in the step C is specifically performed by: removing the phosphate buffer solution, adding fresh phosphate buffer solution, soaking for more than 5min, and repeating for more than 3 times.
7. The method for detecting the morphology of the dendritic spines in the neurons in the brain section according to any one of claims 1 to 6, wherein the standing incubation time in the step C is 30 to 45 min.
8. The method for detecting the morphology of the dendritic spines of the neurons in the brain slice according to any one of claims 1 to 7, wherein the rinsing in the step D is specifically performed by: washing brain slice with phosphate buffer solution for more than 3 times, each time for more than 5min, and washing away from light.
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