CN107418935B

CN107418935B - Method for preparing NOS neuron sample in intestinal parietal plexus of domestic pigeon

Info

Publication number: CN107418935B
Application number: CN201710707968.1A
Authority: CN
Inventors: 孙海基; 马晓丽; 赵盼; 王天霞
Original assignee: JINAN CENTER HOSPITAL; Shandong Normal University
Current assignee: JINAN CENTER HOSPITAL; Shandong Normal University
Priority date: 2017-08-17
Filing date: 2017-08-17
Publication date: 2020-07-31
Anticipated expiration: 2037-08-17
Also published as: CN107418935A

Abstract

The invention relates to a method for preparing a NOS neuron sample in a nerve plexus between intestinal wall muscles of a pigeon, which comprises the following steps: (1) preparing an intestinal tract to be observed; (2) taking an intestinal tract to be observed, and preparing a fixed sample; (3) after the fixed sample is sectioned, stripping a muscle layer, then stripping longitudinal muscles on a nerve plexus, and placing the muscle layer in a phosphate buffer solution for storage to obtain a sample to be dyed; (4) washing a sample to be dyed by a phosphate buffer solution, immersing the sample into a closed solution for warm closing, then immersing the sample into a primary antibody solution, and incubating at room temperature to prepare a primary antibody sample; (5) immersing the primary antibody sample in a goat anti-rabbit IgG solution, incubating in a shaking table, developing in dark, dehydrating, and sealing. The invention adopts the intestinal wall full-layer tablet-NOS immunohistochemical technology to prepare the NOS neuron sample in the intestinal wall intermuscular plexus of the pigeon for the first time, and the separation of the ring muscle and the longitudinal muscle is realized easily by injecting muscle relaxing medicine and stripping the longitudinal muscle before preparing the sample in the preparation process.

Description

Method for preparing NOS neuron sample in intestinal parietal plexus of domestic pigeon

Technical Field

The invention relates to a method for preparing a Nitric Oxide Synthase (NOS) neuron sample in a nerve plexus between intestinal wall muscles of a pigeon, belonging to the technical field of biotechnology.

Background

The gastrointestinal tract peristalsis is mainly caused and depends on smooth muscle contraction, and smooth muscle excitation is mainly mediated by excitatory transmitters such as acetylcholine and the like, and inhibition is mainly mediated by inhibitory transmitters such as Nitric Oxide (NO), etc. in 1990, Bult firstly proposes that Nitric Oxide Synthase (NOS) can be released to catalyze L-arginine to generate NO in vivo when gastrointestinal tract nitrogen neurons are excited, and the NOS is a marker enzyme for NO generation.

The full-layer gastrointestinal slice laying technology is a basic method for researching the structure and the function of a gastrointestinal nervous system, can display the distribution mode of intestinal neurons in submucosa and intersomatic layer in a three-dimensional and visual way, and is widely accepted and applied by the method for researching the intestinal nervous system by utilizing the full-layer gastrointestinal slice laying technology. However, previous studies have focused mainly on mammals, and only a small percentage of the literature has studied the ultrastructure of the chicken enteric nervous system. The form and characteristics of intestinal NOS neurons and the distribution characteristics of different intestinal segments of the domestic pigeon serving as a typical representative of birds are not researched yet.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for preparing an NOS neuron sample in a nerve plexus between intestinal wall muscles of a pigeon.

The technical scheme of the invention is as follows:

a method for preparing a NOS neuron sample in a nerve plexus between intestinal wall muscles of a pigeon comprises the following steps:

(1) injecting a muscle relaxant into the domestic pigeon according to 0.04-0.06 mg/kg, killing the domestic pigeon after 5-30 min, taking the intestinal tract of the domestic pigeon, and cleaning the content in the intestinal tract to prepare the intestinal tract to be observed;

(2) taking the intestinal tract to be observed prepared in the step (1), ligating one end of the small intestine, perfusing the intestinal tract with a phosphate buffer solution containing 3.6-4.4% of paraformaldehyde by mass percent, ligating the other end of the small intestine, placing the intestinal tract in the phosphate buffer solution containing 3.6-4.4% of paraformaldehyde by mass percent for fixing for 6-8 hours at 4 ℃, transferring the intestinal tract to a phosphate buffer solution containing 200-350 g/L of sucrose, and dehydrating overnight at 4 ℃ to prepare a fixed sample;

(3) cutting the fixed sample prepared in the step (2), stripping a muscular layer, laying a serosal surface of the muscular layer on a glass slide in a downward mode, stripping longitudinal muscles on a nerve plexus, reserving an annular muscular layer, and placing the muscular layer in a phosphate buffer solution for preservation to prepare a sample to be dyed;

(4) washing the sample to be dyed prepared in the step (3) by using a phosphate buffer solution, and placing the sample to be dyed in H with the mass concentration of 2.5-3.5% at room temperature₂O₂Soaking for 25-35 min, washing by using a phosphate buffer solution, immersing in a closed solution for sealing at a room temperature for 0.8-1.2 h, immersing in a primary antibody solution with the mass percentage concentration of 0.2-0.5%, standing overnight at 4 ℃, incubating at room temperature for 0.8-1.2 h, and washing by using PBST to prepare a primary antibody sample;

the primary antibody is a rabbit anti-NOS antibody; purchased from bosch de bio;

the confining liquid is a solution containing 5 ml of goat serum per hundred ml of PBST solution;

(5) and (3) immersing the primary antibody sample prepared in the step (4) into a solution containing goat anti-rabbit IgG with the mass percentage concentration of 0.1%, incubating for 1.5-2.5 h by using a shaking table, washing with a PBST solution and a PBS solution respectively, developing for 8-12 min in a DAB solution in a dark place, washing with PBS, dehydrating, and sealing to obtain the goat anti-rabbit IgG.

According to the invention, in the step (1), the weight of the pigeon is preferably 300-340 g.

Preferably, in step (1), the injection amount of the muscle relaxant is 0.05 mg/kg.

Preferably, in step (1), the intestinal contents are cleaned at a concentration of 10^-4And washing the mixture by using a phosphate buffer solution with the mass concentration of 0.75% of mol/L nimodipine.

Preferably, in step (2), the small intestine is an anatomically isolated small intestine.

According to the present invention, in the step (2), the phosphate buffer is preferably a phosphate buffer containing paraformaldehyde at a concentration of 4% by mass.

Preferably, in step (3), the segments are segments of intestine cut to a length of 1 cm.

Preferably, in step (3), the muscle layer is stripped as follows:

the segment of intestine is sleeved on the glass rod, the pin is used for scratching along the longitudinal axis of the intestine, and the ophthalmological forceps gently strip the muscular layer along the scratched position.

According to the present invention, in the steps (2) and (3), the phosphate buffer is preferably a phosphate buffer with a mass concentration of 0.75%.

According to the invention, in the step (4), the washing times are 3 times, and each washing time is 5-15 min.

According to the invention, in the step (5), the PBST solution is washed for 4 times, and the washing time is 5-15 min each time; the washing frequency of the PBS solution for the first time is 1 time, and the washing time is 5-15 min; the washing times of the second PBS solution are 3 times, and the washing time is 5-15 min each time.

According to the present invention, in the step (5), the dehydration is performed by stepwise dehydration using 75%, 95% and 100% alcohol by volume, respectively, and then replacing the alcohol with xylene.

According to the invention, in the step (5), the sealing piece is a sealing piece made of neutral gum.

The PBST solution, the PBS solution and the DAB solution are all common commercial products in the field.

Advantageous effects

1. The method adopts the intestinal wall full-layer sheet-laying-NOS immunohistochemical technology to prepare the NOS neuron sample in the intestinal wall intermuscular plexus of the pigeon for the first time, and the separation of the ring-shaped muscle and the longitudinal muscle is realized easily by injecting muscle relaxing medicine and stripping the longitudinal muscle before preparing the sample in the preparation process; the problem that due to the fact that the intestinal wall of a pigeon is thick, the combination of the ring muscle and the longitudinal muscle is tight and difficult to peel off, the dyeing and observation of NOS neurons in the nerve plexus are affected by the non-peeled muscle fibers is solved; thereby obtaining a sample which can clearly observe NOS neurons in the intramuscular plexus of the enteric nervous system of the pigeon;

2. because of the different osmotic pressure of bird cell and mammal cell, the invention adopts PBS buffer solution with salt concentration of 0.75%, can prevent the influence on cell morphology during the process of treating tissue.

Drawings

FIG. 1 is a 150-fold magnification morphological observation photograph of NOS positive neurons of the myenteric wall muscle plexus of a pigeon;

in the figure: A. duodenum, arrows show protrusions on NOS-positive neuronal cells; B. jejunum; C. the ileum; D. the cecum; E. a rectum;

FIG. 2 is a photograph showing the observation of the morphology of the ganglion of the myenteric plexus of domestic pigeon at 25 times magnification;

in the figure: A. the duodenum; B. jejunum; C. the ileum; D. the cecum; E. a rectum;

FIG. 3 is a photograph of morphological observation of NOS positive ganglion size and third plexus at 100 times magnification;

in the figure: A. large ganglia; B. middle and small ganglia; C. tertiary plexus, primary nerve fibers (P), secondary nerve fibers (S), tertiary nerve fibers (T);

FIG. 4, photographs of morphology observation with and without muscle relaxant treatment;

in the figure: A. group without muscle relaxant treatment; B. groups were treated with muscle relaxant.

Detailed Description

The technical solution of the present invention is further described with reference to the following examples, but the scope of the present invention is not limited thereto.

Sources of materials

Healthy adult domestic pigeons are purchased from the animal experiment research center of Shandong university and have the weight of 300-340 g;

muscle relaxant was purchased from Shanghai Jingdu Biotechnology Co., Ltd;

PBST solution, PBS solution and DAB solution were purchased from China fir Jinqiao biotechnology, Inc. in Beijing;

goat anti-rabbit IgG was purchased from Kyoto Chimona Jinqiao Biotechnology, Inc. of Beijing;

rabbit anti-NOS antibodies were purchased from Boshide Biometrics;

examples

1 materials and methods

1.1 Experimental animals

Healthy adult domestic pigeons are not limited in sex, purchased by animal experiment research center of Shandong university, 10 in number and 300-340 g in weight.

1.2 taking materials

After injecting muscle relaxant, the head of the domestic pigeon is immersed in water and killed by suffocation; after the pigeon is sacrificed, the abdomen of the pigeon is upwards placed in an anatomical disc, the abdomen feather of the pigeon close to the cloaca is soaked by clear water and then pulled out, the skin is transversely cut by an anatomical scissors, the abdominal cavity is rapidly opened, the whole intestinal tract is separated along the mesentery, and the duodenum, jejunum, ileum, cecum and rectum are separated according to anatomical positions for later use.

1.3 making tear tabs

1.3.1 fixation

With newly prepared nimodipine (10)^-4mol/L), ligating one end of the small intestine, perfusing and filling the small intestine to the maximum extent with 4% paraformaldehyde fixed solution phosphate buffer solution (pH7.4), ligating the other end, placing the small intestine in 4% paraformaldehyde fixed solution for fixing for 6-8 h (4 ℃), transferring the small intestine into 300 g/L sugarcane phosphate buffer solution for dewatering overnight (4 ℃).

1.3.2 tear tabs

Cutting the fixed intestinal canal into 1cm long segments, sleeving on a proper glass rod, scratching the longitudinal axis of the intestinal canal by using a pin, slightly stripping a muscular layer along the scratched position by using an ophthalmic forceps, flatly paving the stripped muscular layer serosa on a glass slide in a downward direction, stripping longitudinal muscles on a nerve plexus by using forceps under a dissecting microscope, reserving a ring muscular layer (the ring muscle of the intestinal wall muscular layer of birds is outside, and the longitudinal muscles are inside), preparing an intersomatic nerve plexus between the longitudinal muscles and the ring muscles, and placing the intersomatic nerve plexus in 0.01 mol/L Phosphate Buffer Solution (PBS) for dyeing.

1.4 immunohistochemical technique for detecting expression of NOS Positive neurons

Washing the prepared sample to be dyed with 0.75% phosphate buffer solution for three times, and standing at room temperature in 3% H solution after 5 minutes each time₂O₂Washing for 30min, washing for three times by using a phosphate buffer solution with the mass concentration of 0.75%, soaking in a closed solution for warm sealing for 1h after 10 minutes each time, then soaking in a primary antibody solution with the mass percentage concentration of 0.3%, standing overnight at 4 ℃, then incubating for 1h at room temperature, washing for three times by using PBST (basic-bound-substance transport system) and washing for 15 minutes each time to prepare a primary antibody sample;

the primary antibody is a rabbit anti-NOS antibody;

then, the prepared primary antibody sample is immersed in a solution containing goat anti-rabbit IgG with the mass percentage concentration of 0.1%, incubated for 2h by a shaker, washed with PBST solution four times for 10 minutes each time, then washed with phosphate buffer solution with the mass concentration of 0.75% for 10 minutes, developed for 10 minutes in a DAB solution in a dark place, washed with PBS three times for 10 minutes each time, and a developed section is prepared.

1.5 sealing sheet

And (3) sucking the developed slices into a culture dish containing phosphate buffer solution with the mass concentration of 0.75%, sequentially sticking the slices onto glass slides by using a brush pen, and naturally drying the glass slides. After that, alcohol stepwise dehydration (75% alcohol, 95% alcohol, 100% alcohol (two steps), respectively, in volume percentage concentration) and xylene (two steps) were performed for transparency. When xylene is not completely volatilized, the neutral gum is encapsulated.

1.6 statistics and analysis

The specimens were first scanned one by one with a panoramic midi automated digital scanner and the pictures taken were then imported into Caseviewer software (version 6.0) for quantitative analysis. The immunohistochemical staining is positive if the staining is dark brown yellow, weak positive if the staining is light brown yellow, and negative if the cell body has the same color as the background.

Then selecting a specimen with good dyeing, amplifying by 4 times, and randomly selecting a specimen with an area of 10.0mm²Counting the number of ganglia, counting more than 10 slices, counting more than 10 times in different areas of each slice, and calculating the density of NOS positive ganglia. Amplifying by 10 times, randomly selecting area of 5.0mm²And (3) counting the number of the NOS positive neurons, counting more than 10 slices, counting more than 10 times in different areas of each slice, and calculating the density of the NOS positive neurons. Amplifying by 700 times, counting the perimeter and the area of the cell body and the cell nucleus of the NOS positive neuron, counting more than 10 slices, continuously counting more than 20 slices per slice, and calculating the size of the cell body and the cell nucleus of the NOS positive neuron in the internus fascicular plexus.

Finally, One-Way variance (One-Way-ANOVA) analysis was performed using SPSS 20.0 statistical software (pasvstattics 20), and data were expressed as Mean soil standard deviation (Mean soil SD).

2 results

2.1 Pigeon gut wall intermyoplexus NOS Positive neuron morphology and distribution

NOS immunohistochemical staining shows that NOS positive neurons in intestinal muscles of domestic pigeons are different in size, cytoplasm staining is brownish yellow with different intensities, and nuclei are not stained. NOS positive neurons vary in shape, and the neuron cell bodies are usually oval, circular, fusiform, and the like (see FIG. 1). The neuron nucleus is larger, is inclined to one side and is not colored, the cytoplasm is not colored in different depths, the NOS positive neurons of duodenum, jejunum and ileum are colored in deeper depth, the rectum is inferior, and the caecum is colored in the lightest depth. The majority of NOS positive neurons are distributed within the ganglia and the minority is distributed in internodal nerve fiber bundles.

In the duodenum, the jejunum, the NOS neurons bodies are mostly between 16 μm and 20 μm in diameter, and at high power, many bodies can be seen with short protrusions or a flattened long protrusion, which are generally oval, with fusiform, comet-like, triangular, or irregular shapes (see fig. 1-a). In the cecal section, there are occasional long processes with long projections, comet-shaped, while most cells are oval or round (see FIGS. 1-D, 1-E).

2.2 Density of NOS Positive neurons in the intestinal plexus of Pigeon

The density of NOS positive neurons in different intestinal segments differed greatly (as shown in FIG. 2). The number of NOS positive neurons in the duodenal, jejunal, ileum, cecum and rectus myenteric plexus is respectively as follows: 5.78 +/-2.14 pieces/mm²33.20 + -3.24 pieces/mm²14.80 +/-3.29 pieces/mm²13.30 +/-5.54 pieces/mm²And 23.33. + -. 2.23/mm². NOS positive cells were found to have the highest density in the jejunum, the next rectum, the ileum and caecum were substantially similar, and the number of duodenum was the lowest (as shown in Table 1).

TABLE 1 number of NOS Positive neurons in the internus musculus plexus of the intestinal tract (pieces/mm)²)

In contrast to the duodenum, a means P < 0.01; b represents P <0.01 compared to jejunum; c denotes P <0.01 compared to ileum; in contrast to the cecum, d denotes P < 0.01.

2.3 size of enteric canal Mylar plexus NOS Positive neuron of domestic Pigeon

The sizes of the NOS positive neurons of the nerve plexus between different intestinal segments are greatly different, wherein the cell bodies of the NOS positive neurons of the duodenal segments have the largest perimeter and area, the next ileum and the smallest cecum. The duodenum segments ileum, caecum and rectum are significantly larger, less ileal, than the jejunum, ileum, caecum and rectum, and the ileum, caecum and rectum are substantially similar in circumference and area. (see Table 2)

TABLE 2 comparison of sizes of NOS neurons in different intestinal segments of domestic pigeon (circumference: μm area: μm)²)

In contrast to the duodenum, a means P < 0.01; b represents P <0.01 compared to jejunum; c denotes P <0.01 compared to ileum; in contrast to the cecum, d denotes P <0.01

2.4 Pigeon myenteric plexus ganglion morphology

The full-layer intestinal wall tablet-NOS immunohistochemical technique can clearly show the structural form of the myenteric plexus of each segment of the pigeon and the form and density of the ganglion. The domestic pigeon duodenum ganglia are obvious, mostly in the shape of star, butterfly and long fusiform, long protrusions are emitted by the positive neurons to form internode nerve bundles, the ganglia and the internode nerve bundles form a regular pentagon, and finally the ganglia and the internode nerve bundles are connected into a nerve network with a regular shape (see fig. 2A). The morphology of the ganglia and plexuses of the ileum and ileum segments is similar to that of the duodenum (see fig. 2B, C). Compared with the small intestine section, the ganglion of the caecum section has obviously small volume and unobvious shape. Internodal nerve bundles are thicker and connected to form an irregular network-like structure (see fig. 2D). The ganglion of the rectal segment is mostly in the shape of a star and a triangle, and the internode nerve bundles are short and thick. The nerve bundles and ganglia are interleaved to form a "cellular" neural network (see fig. 2E). The network consisting of ganglia and internode nerve fiber bundles is called the Primary internuscular plexus (Primary nerve fibers). The primary nerve fibers initiate branches, forming secondary (secondary neural fibers) and Tertiary (Tertiary neural fibers) plexuses. (see FIG. 3C)

In different intestinal segments, the positive ganglia can be roughly classified into the following categories according to the number of NOS positive neurons, the area and morphology of the positive ganglia, and the like: (see FIG. 3A, FIG. 3B)

1. Large positive ganglia: total number of NOS positive neurons is more than 50, and area is more than 0.1mm²And are mostly star-shaped, long spindle-shaped, etc. (see FIG. 3-A).

2. Medium positive ganglia: the total number of NOS positive neurons is 20-50, and the area is 0.05mm²～0.1mm²And the other is long fusiform, triangular, quadrilateral, butterfly-shaped and the like (see fig. 3B).

3. Small positive ganglia: total number of NOS positive neurons is less than 20, and area is less than 0.05mm²Most of them are triangles, and some are thickened areas of nerve fibers. (FIG. 3B).

The large ganglia and the small ganglia are fewer in number, most of them are medium ganglia, and all the segments of intestines are similar.

2.5 Pigeon myenteric plexus ganglion Density

The density of NOS positive ganglia in different intestinal segments differed greatly (fig. 3C). The number of ganglia per 10mm in the duodenal, jejunal, ileum, cecum and rectus myenteric plexus²Respectively as follows: 4.00 + -1.00, 8.55 + -1.51, 15.33 + -3.00, 24.89 + -1.05 and 27.22 + -1.39. NOS positive ganglia are most dense in the rectum, followed by the caecum, ileum and jejunum, and least in the duodenum, as detailed in table 3:

TABLE 3 number of ganglia in different intestine segments of pigeon (unit: number/10 mm)²)

Comparative example 1

Samples of NOS neurons in the intestinal parietal plexus of domestic pigeons were prepared as described in the examples, except that no muscle relaxant was injected. The results are shown in FIG. 4A.

As can be seen in fig. 4, in the muscle relaxant-untreated group of fig. 4A, there were more muscle fibers in the plating, resulting in less staining of NOS positive neurons and less distinguishing; fig. 4B shows that the fibers in the plating muscle were peeled off more cleanly, the interspinal plexus was more prominent, and NOS positive neurons were stained more deeply and easily distinguished after the treatment with the muscle relaxant.

Comparative analysis

The method is characterized in that the nerve plexus is a flat net structure, and the full-sheet technology and immunohistochemical staining are the best methods for observing the shape of the nerve plexus between the intestinal wall longitudinal muscle layer and the circular muscle layer, the shape and the shape of ganglia, the density of neurons and the like, Nitric Oxide Synthase (NOS) is used as a marker enzyme of NO and catalyzes L arginine to generate NO in vivo, and the nitric oxide synthase is proved to be NADPH-diaphorase (NDP), NDP histochemistry can be better used for positioning research of NOS.

The samples prepared by the intestinal wall full-layer slice laying and NOS immunohistochemical technology can clearly show that NOS positive neurons and positive nerve fibers are widely distributed in each intestinal segment muscle layer of the pigeon, and the bundled positive nerve fibers are repeatedly branched to form a secondary network and a tertiary network. The structures of the positive plexus of duodenum, jejunum and ileum of the domestic pigeon are similar, and the shapes are clear and regular. Moreover, the ganglia are obvious, and are mostly in the shapes of stars, butterfly shapes and long fusiform shapes. Compared with the small intestine section, the caecum section of the pigeon has obviously small volume and unobvious shape. The ganglion of the rectal segment is in a star shape and has a relatively large volume. Suggesting that the shapes of the small intestine, the caecum and the rectal ganglion of the domestic pigeon are greatly different. The number of ganglia, the density of duodenum, jejunum, ileum, caecum and rectal ganglia, increased gradually and differed significantly. This is in accordance with the current findings that adult chickens have a rule that the myenteric plexus NOS positive ganglia are the most rectal and the least dodecarectal.

The total intestinal wall layer tablet-laying and NOS immunohistochemical method shows that different intestinal section NOS positive neurons are differently stained, the duodenum, jejunum and ileum NOS positive neurons are more deeply stained, the rectum positive neuron cytoplasm is more slightly stained, the difference is larger, and most of cecal neurons are more lightly stained. The different staining intensity of positive neurons in the gastrointestinal tract may be related to the different functional state, synthesized or stored content of the cells. Cserni and the like speculate that NOS positive neurons are deeply dyed and have higher NOS content and possibly are inhibitory motor neurons; the less stained may be interneurons, sensory neurons or glial cells. The inventor believes that the difference of the positive neuron staining depth of different intestine sections of domestic pigeon probably indicates that the nitric oxide synthase content in NOS positive neurons of small intestine, colon and rectal intestinal wall of the pigeon is greatly different, the small intestine content is the highest, the caecum content is the lowest, and the NOS neurons can exist in different subgroups in the intestinal tract of the domestic pigeon. The total intestinal wall layer is laid on a sample treated by combining a NOS immunohistochemistry method, and through observation, the pigeon NOS positive cells are observed to have the highest density in the jejunum, the second rectum, the ileum and the caecum are basically similar, and the number of the duodenum is the least. Therefore, the sample has very high definition and the like, and can clearly observe that NOS positive neuron cells have different sizes and different forms, cytoplasm staining is brownish yellow with different intensities, and nuclei are not stained. The branches from the primary nerve fiber can be clearly distinguished to form the secondary and tertiary nerve plexuses.

The inventor selects an automatic digital scanner to scan and photograph the sample one by one, enlarges the picture by 700 times by Caseviewer software (version 6.0), and measures the neuron cell volume area and nucleus area by a cell image analysis method so as to analyze the change of the intestinal neuron form.

The samples treated by the method can clearly observe that abundant NOS positive neurons are distributed in the intramuscular nerve plexus of the enteric nervous system of the pigeon, the dyeing depth of the NOS positive neurons in duodenum, ileum, jejunum, caecum and rectum of the pigeon is light, and the form and the density of the neurons are greatly different. Meanwhile, the method can display the shapes and densities of the ganglia and the plexuses of different intestine sections of the domestic pigeon. Therefore, the intestinal wall full-sheet combined NOS immunohistochemistry method can be used as a first-choice experimental technology for displaying the complete appearance of the nerve plexus between pigeon muscles.

Claims

1. A method for preparing a NOS neuron sample in a nerve plexus between intestinal wall muscles of a pigeon is characterized by comprising the following steps:

the weight of the domestic pigeon is 300-340 g;

the phosphate buffer solution is a phosphate buffer solution containing 4 mass percent concentration of paraformaldehyde, and the phosphate buffer solution is a phosphate buffer solution with 0.75 mass percent concentration;

the sections are intestinal sections cut into lengths of 1 cm; the muscle layer stripping step is as follows:

sleeving the intestinal section on a glass rod, scratching the intestinal tube along the longitudinal axis of the intestinal tube by using a pin, and slightly stripping a muscular layer by using the ophthalmic forceps along the scratched position;

the phosphate buffer solution is 0.75 mass percent

the primary antibody is a rabbit anti-NOS antibody;

(5) immersing the primary antibody sample prepared in the step (4) into a solution containing goat anti-rabbit IgG with the mass percentage concentration of 0.1%, incubating for 1.5-2.5 h by using a shaking table, washing with a PBST solution and a PBS solution respectively, developing for 8-12 min in a DAB solution in a dark place, washing with PBS, dehydrating, and sealing to obtain the product;

the dehydration is to gradually dehydrate by adopting 75 percent, 95 percent and 100 percent alcohol by volume percentage concentration and then replace the alcohol by dimethylbenzene.

2. The method of claim 1, wherein in step (1), the muscle relaxant is injected in an amount of 0.05 mg/kg.

3. The method of claim 1, wherein in step (1), the intestinal contents are cleaned at a concentration of 10%^-4And washing the mixture by using a phosphate buffer solution with the mass concentration of 0.75% of mol/L nimodipine.

4. The method of claim 1, wherein in step (2), the small intestine is an anatomically isolated small intestine.

5. The method according to claim 1, wherein in the step (4), the washing times are 3 times, and each washing time is 5-15 min.

6. The method according to claim 1, wherein in the step (5), the PBST solution is washed for 4 times, and each washing time is 5-15 min; the washing frequency of the PBS solution for the first time is 1 time, and the washing time is 5-15 min; the washing times of the second PBS solution are 3 times, and the washing time is 5-15 min each time.

7. The method of claim 1, wherein in step (5), the mounting is by using a neutral gum mounting.