CN112444436B

CN112444436B - Pretreatment method for integral immunostaining of adult diaphragm

Info

Publication number: CN112444436B
Application number: CN202011324628.9A
Authority: CN
Inventors: 沈承勇; 杜温佑; 许文涛; 张克兢
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2020-11-23
Filing date: 2020-11-23
Publication date: 2022-06-21
Anticipated expiration: 2040-11-23
Also published as: CN112444436A

Abstract

The invention discloses a pretreatment method for integral immunostaining of an adult diaphragm, which is characterized in that the diaphragm is clamped between two organic glass sheets, so that the space between muscle fibers of the adult diaphragm is enlarged, connective tissues are easy to cut off, the pressed diaphragm is uniformly expanded, the diaphragm still keeps flat and expanded even after a device is removed, the pressure does not damage the diaphragm, the thickness of the diaphragm is thinned, the integral immunostaining of the adult diaphragm is facilitated, the background color of an immunostained image is lighter, and the image is easier to observe.

Description

Pretreatment method for integral immunostaining of adult diaphragm

Technical Field

The invention particularly relates to a pretreatment method for integral immunostaining of an adult diaphragm.

Background

Neuromuscular junction (NMJ) is a synaptic junction whose three component structures are motor neurons, muscle fibers and peripheral schwann cells (Darabid et al 2014, Li et al 2018; Wu et al 2010). Morphological changes in NMJ are hallmarks of many neuromuscular diseases, such as the characteristic Amyotrophic Lateral Sclerosis (ALS), Spinal Muscular Atrophy (SMA) and Muscular Dystrophy (MD) (Fischer et al). Pathological features such as axonal end-loss innervation, swelling, multiple innervation, loss of NMJ complexity, NMJ fragmentation, etc. are well understood. These are all easily recognized by immunostaining, effectively indicating synaptic function.

In a mouse model, some thin, flat muscles (such as the diaphragm, the transverse abdominus, the deltoid and the cranial) were stained globally (Murray et al, 2010). Observations of the overall innervation pattern allow unbiased assessment of pathophysiological changes, and motor unit size, axonal branching, and selective vulnerability to disease are well described (Banks et al, 2003). Among these flat muscles, the understanding of NMJ is of particular interest, since diaphragm dysfunction is closely related to respiratory function. In fact, dyspnea in motor neuron disease (such as ALS) patients can be assessed by diaphragm weakness, e.g., evoked responses to transcutaneous phrenic nerve stimulation may predict hypoventilation and survival in ALS patients (Pinto et al, 2009; Pinto et al, 2012). The pathology of diaphragm NMJ loss is very important and many studies use it to examine NMJ development because it is flat and readily available, particularly at the embryonic or neonatal stage.

However, the understanding of the structural pattern of intact NMJ in adulthood remains limited, as the diaphragm becomes thicker and covered by dense connective tissue (Sefton et al, 2018), resulting in poor antibody permeability and less susceptibility to immunostaining.

Disclosure of Invention

Aiming at the situation, the invention provides a pretreatment method for integral immunostaining of an adult diaphragm, which aims to overcome the defects of the prior art.

In order to achieve the purpose, the invention provides the following technical scheme:

a pretreatment method for integral immunostaining of an adult diaphragm comprises the following steps:

(1) flatly paving the dissected diaphragm on a flat plate to expand the diaphragm;

(2) 1ml of PBS buffer solution (pH7.4, 4%) paraformaldehyde was dropped onto the other plate;

(3) placing the flat plate attached with the diaphragm in the step (1) on the flat plate obtained in the step (2), wherein the diaphragm is positioned between the two flat plates, and screwing bolts and nuts in a crisscross sequence until the flat plate can not be screwed again;

(4) keeping the whole device obtained in the step (3) at 4 ℃ for 20 minutes, and fixing the diaphragm;

(5) taking out the diaphragm from the flat plate, and placing the diaphragm in a container filled with PBS buffer solution;

(6) removing connective tissue from the surface of the diaphragm;

(7) after the connective tissue is removed, the diaphragm is clamped between the two plates again, and the bolts and nuts are tightened in a criss-cross sequence; until the screw can not be screwed down any more; holding at 4 deg.C for 20 min;

(8) then taking the diaphragm out of the device, and soaking the diaphragm into 4% paraformaldehyde PBS buffer solution with pH7.4 at 4 ℃ for 20 minutes;

(9) the diaphragm was then washed with PBS buffer containing 0.1% glycine for 10 minutes at room temperature.

Further, in the step (1) and the step (2), the flat plate has at least one flat surface, and each flat plate is provided with at least two through holes.

Further, in step (1), the flat surface of the plate is contacted with an adult diaphragm.

Further, in the step (1) and the step (2), the thickness of the flat plate is greater than or equal to 6 mm.

The invention has the beneficial effects that:

(1) according to the invention, the diaphragm is clamped between the two organic glass sheets, so that the space between muscle fibers of the adult diaphragm is enlarged, connective tissues are easy to cut, the pressed diaphragm is uniformly expanded, the diaphragm is still flat and expanded even after the device is removed, the pressure does not damage the diaphragm, the thickness of the diaphragm is thinned, the integral immunostaining of the adult diaphragm is facilitated, the background color of the immunostaining image is lighter, and the image is easier to observe.

(2) The pretreatment method is adopted to treat the diaphragm, then the adult diaphragm is integrally immunostained, and the details of morphological characteristics can be identified in the dyed image, so that the reliability of evaluating the phenotype of development and degenerative neuromuscular can be improved. The pretreatment method is rapid and simple, and is convenient for providing a comprehensive view of NMJ distribution, wherein the comprehensive view contains complete detailed information of single NMJ.

(3) The present invention provides a convenient and versatile method to improve the visualization of innervation networks, not only for immunolabeling, but also for classical esterase silver staining.

(4) The existing tissue transparency technologies, such as 3DISCO/iDISCO and CUBIC, are the most advanced technologies, but a plurality of time-consuming steps (more than 10 days in total) are needed before imaging, when flat muscles such as diaphragm are researched, the image details are not improved, the pretreatment method can be successfully completed within 2 days based on the conventional immunostaining procedure by adopting the pretreatment method, and the pretreatment method can help antibody penetration without removing the fascia.

(5) The pretreatment method of the invention can effectively stretch the diaphragm muscle and keep the integrity of the whole structure; the entire innervated structure (labeled with anti-NF and anti-SV 2) was clearly immunostained for easy observation, facilitating analysis of innervation of the diaphragm, while retaining different types of NMJ structural features.

Drawings

Figure 1 is a schematic illustration of the preparation of the diaphragm by muscle stretching; wherein (a) is a schematic diagram of a device for pressing the diaphragm, (B) is a schematic diagram of the diaphragm on plexiglass, (C) is a schematic diagram of the diaphragm flattened and spread between two plexiglass sheets after the device is closed, (D) is a schematic diagram of the diaphragm remaining flat after the device is removed, (E) is a statistical result diagram of the length (ventral side to dorsal side) and width (widest distance in the semi-diaphragm) of the pressed and uncompressed diaphragm of a 2.5 month old male mouse, (F) is a statistical result diagram of the area of the pressed and uncompressed diaphragm, (G) is a statistical result diagram of the thickness of the pressed and uncompressed diaphragm, (H) is a comparative result diagram of the extension of muscle fibers, and the actin filaments are labeled phallodin-594, phallodin (red), DAPI (blue).

FIG. 2 is a schematic representation of NMJs in adult mouse diaphragm, (A) is a representative immunostaining image of uncompressed adult diaphragm; the enlarged views of the areas in boxes 1-3 are arranged sequentially from left to right, with the dense parallel green lines (the white arrows indicate the parallel green lines) being the autofluorescence signals emitted by the muscle fibers; NF/SV2 (green), BTX (red); (B) is a typical immunostaining image of stressed adult diaphragm, with innervated NMJs shown in yellow, with enlarged views of the area in boxes 1-3 in order from left to right, NF/SV2 (green), BTX (red), (C) is a statistical plot of the area after the non-stressed and stressed diaphragms, and (D) is a statistical plot of fragmentation index, (E) is a representative plot of the acetylcholinesterase silver method treated non-stressed adult diaphragm, with enlarged views of the area in boxes 1-2 in order from top to bottom, (F) is a representative image of the acetylcholinesterase silver method treated stressed adult diaphragm, axons (dark brown); the enlarged views of the regions in the end plate (indigo), boxes 1-2 are arranged in sequence from top to bottom.

FIG. 3 shows SOD1^G93AMorphological changes in pathological NMJs in mice, where (A) is the end stage of disease (4 months old), SOD1^G93ARepresentative image of mouse diaphragm, bulk NMJS denervated (white arrow), enlarged views of the area in boxes 1-3, arranged sequentially from left to right, (B) age-matched control wild-type and SOD1^G93AStatistics of postsynaptic areas of mouse diaphragms, analyzing 90 end plates of each diaphragm, and analyzing 3 mice in each group without significance, (C) fragmentation index statistics, analyzing 90 end plates of each diaphragm, and analyzing 3 mice in each group without significance, (D) neural occupancy statistics, analyzing 1000 end plates of each diaphragm, and analyzing 3 mice in each group, (E) high power confocal images of NMJs, and WT represents high power confocal images of NMJs of control wild type mouse diaphragms; a plurality of innervated NMJS (white arrow a), partially innervated NMJS (blue arrow b); NMJS completely denervated (purple arrow c), NF/SV2 (green), BTX (red).

Figure 4 is a representative immunostaining image of an enlarged version of the compressed diaphragm of figure 2B, NF/SV2 (green), BTX (red).

Figure 5 is a schematic of bulk immunostaining of the diaphragm with three different fluorophores, NF (green), SV2 (blue), BTX (red).

FIG. 6 is a sample stained 6 months ago in FIG. 2B, a re-acquired image, NF/SV2 (green), BTX (red).

Detailed Description

The technical solutions of the present invention are further described in detail below with reference to the accompanying drawings, and it should be noted that the detailed description is only for describing the present invention, and should not be construed as limiting the present invention. The reagents and/or devices used in the following examples are commercially available. The PBS buffer solutions described in the following examples are all 0.1mol/L, pH7.4 PBS buffer solution, 1M ═ 1 mol/L.

Example 1

A device for pre-treatment of integral immunostaining of adult diaphragm comprises at least two flat plates as shown in figure 1A, wherein at least two flat plates are respectively provided with at least one flat surface, and each flat plate is provided with at least two through holes.

In use, the flat surface of the plate is brought into contact with the adult diaphragm.

Generally, a 5cm by 5cm plate is suitable for the diaphragm of all adult mice. The size of the flat plate can be adjusted correspondingly according to the size of the diaphragm of the animal, and the size of the flat plate is not limited by the invention.

In some preferred forms, the plate may take a variety of shapes, such as rectangular, circular, diamond, other polygonal shapes, etc., whatever the shape, it is desirable to ensure that the plate covers the diaphragm without exposing the diaphragm to the plate.

In some preferred modes, the through holes may be screw holes or smooth holes, and the invention does not limit the pattern of the through holes. The location of the through holes can be determined according to actual conditions, in the embodiment, as shown in fig. 1A, holes are drilled at four corners at a distance of 1cm from the edge, and the distribution can be suitable for various adult diaphragms.

In some preferred modes, the through holes are ensured to be symmetrically arranged, so that the force applied to the diaphragm is more uniform.

In some preferred modes, the thickness of the flat plate is greater than or equal to 6mm, in this embodiment, the thickness of the flat plate is 6mm, and if the thickness of the flat plate is less than 6mm, the bending degree of the flat plate after being pressed is affected, and therefore the uniformity of the pressure is affected.

In some preferred modes, the device further comprises a fastener, and the fastener can be a bolt and a nut, a screw or other fasteners.

In some preferred modes, the flat plate is made of transparent materials, the flat plate can be a polymethyl methacrylate plate, and the flat plate can also be a transparent plastic plate. The flat plate is made of transparent materials, so that the condition that the diaphragm is extruded in the device can be conveniently observed.

Example 2

All mice were kept under standard conditions in the experiment, 12 hours under light and 12 hours in the dark, and food and water were freely available. All animal experimental procedures were approved by the animal protection and use committee of university of zhejiang.

Obtaining a diaphragm: the abdominal skin of the mouse is cut into the abdominal cavity, the sickle ligament is cut off from the diaphragm, the liver and the diaphragm are separated, the foot muscle of the diaphragm is clamped by forceps, the diaphragm is slightly pulled outwards, and the diaphragm is separated. The diaphragm can be dissected along the dorsal wall with an arc dissector, the dissected diaphragm cleaned in PBS to remove blood and trimmed slightly by dissecting the calf muscle and adipose tissue (Sefton et al, 2018). The diaphragm can also be obtained by other methods in the prior art, and the invention does not limit the obtaining method of the diaphragm.

(1) the dissected diaphragm was placed on a plexiglas plate (5 cm. times.5 cm) and gently spread flat to ensure that the muscles did not fold, the device used in this example is shown in figure 1A.

(2) 1ml of buffered solution of Paraformaldehyde (PFA) PBS, pH7.4, 4% (w/v) was dropped onto another plexiglass slide. In this example, a paraformaldehyde solution was dropped onto the center of an organic glass sheet.

(3) Placing the organic glass sheet with the diaphragm on the organic glass sheet dripped with the paraformaldehyde PBS buffer solution, and lightly screwing the bolt and the nut in a crisscross sequence by hand (namely screwing the bolt in a diagonal sequence); further tightening is performed with a screwdriver in the same order until tightening is no longer possible.

(4) The whole device obtained in step (3) was kept at 4 ℃ for 20 minutes to fix the diaphragm. This procedure facilitates the excision of connective tissue and enlarges the gaps between muscle fibers.

(5) The diaphragm was removed from the plexiglass slide and placed in a clean petri dish with PBS. Specifically, the nuts and bolts are removed first, and the pressed diaphragm is carefully removed from the plexiglass slide with forceps and placed in a clean petri dish with PBS. After the bolts and nuts are removed, the diaphragm tissue keeps the shape and the volume almost unchanged (as shown in fig. 1D).

(6) Connective tissue on the diaphragm surface was gently removed with #5 forceps. It is necessary to remove connective tissue from the surface of the diaphragm because this prevents the antibody solution from penetrating the muscle fibers. Care should be taken during cleaning to avoid damaging muscles or nerve fibers.

(7) After removal of the connective tissue, the muscle was again sandwiched between two plexiglass plates (this step was performed as before), and the bolts and nuts were lightly tightened in criss-cross order (i.e., the bolts were tightened in diagonal order); further tightening with a screwdriver in the same order until the screw can no longer be tightened; held at 4 ℃ for 20 minutes for further stretching.

(8) The diaphragm was then removed from the device and immersed in 4% (w/v) paraformaldehyde PBS buffer at pH7.4 for 20 minutes at 4 ℃.

(9) The diaphragm was then washed with PBS buffer (w/v) containing 0.1% glycine at Room Temperature (RT) for 10 minutes.

Non-stressed control group: the diaphragm was secured to the chest (no pressure applied), for 20 minutes, and the connective tissue was removed. Other embodiments are the same as the experimental group.

Cross-diaphragm immunostaining

The diaphragm was incubated in 1ml of blocking buffer, PBS buffer containing 1% Triton-X100(v/v), 2% bovine serum albumin BSA (w/v) and 4% normal goat serum (v/v), for 30 minutes at room temperature with gentle stirring. The diaphragm tissue was transferred to a novel blocking buffer comprising anti-neurofilament and anti-synaptobrevin 2 antibodies, anti-neurofilament NF antibodies (diluted at 1:500, rabbit monoclonal, antibody cat No. 2837S; Cell Signalling Technology, Inc.) and anti-synaptobrevin 2 antibodies (SV2 concentrate, diluted at 1:500, mouse monoclonal, DSHB).

The tissue obtained above was incubated overnight in buffer at 4 ℃, gently shaken, and washed three times with PBS buffer the next day for 30 minutes. The diaphragm muscle was then incubated for 1 hour at room temperature in blocking buffer containing Alexafluor 488-binding antibody (diluted at 1:1000, goat anti-mouse IgG and goat anti-rabbit IgG, Invitrogen) and 2. mu.g/ml alpha-bungarotoxin tetramethylrhodamine (T0195, Sigma) protected from light. All incubation and washing steps were performed in shaking or shaking. It is recommended to check the fluorescence signal of the sample before installation. If the signal is weak or intermittent, the step (3) can be repeated, and the pressing treatment is carried out again to carry out the antibody treatment.

Subsequently, the diaphragm muscle was fixed with water-soluble mounting medium (F4680, Sigma), covered with a lid, and left overnight at 4 ℃ so that the mounting medium coagulated. If long term storage is required, the specimen edges are sealed with clear nail polish. The specimen can be stored for at least 6 months without significant loss of fluorescence intensity (as shown in fig. 2B and 6). To assess muscle damage that may result from stress, the stressed and unstressed diaphragm was stained with Phalloidin-594 (diluted at 1:1000, A12381, Invitrogen) to mark the actin bands for 1 hour at RT conditions at room temperature. The specimens were mounted, covered and stored at 4 ℃ until imaged.

Acetylcholinesterase silver staining of diaphragm

Axonal and endplate staining of the diaphragm was performed (kiennan, 1996). Briefly, in the presence of 0.02M CaCl₂、0.01M K₃[Fe(CN)₆]And 0.01M K₄[Fe(CN)₆]In 0.02M Tris buffer (pH 7.2) for 45 min. The diaphragm was then rinsed twice with water and dehydrated/rehydrated in each solution by a graded ethanol series (70% ethanol, 95% ethanol, 100% ethanol, xylene, 100% ethanol, 95% ethanol, 70% ethanol) for 2 minutes.

Thereafter, the diaphragm was rinsed three times with clear water. Staining axon with 20% silver nitrate solution for 20min, washing with water for three times, and washing with hydroquinone (0.2% hydroquinone at 1% Na)₂SO₃In solution) silver was reduced for 10min until the axons were dark brown or black. After washing with water twice, toning with gold in 0.2% gold chloride solution for 3minAnd (4) contrast. At this point, the axons will be less dark and the development will be at 5% Na by mass₂S₂O₃The solution was stopped for 5 minutes. The samples were mounted and covered as described previously.

Imaging

Red-labeled postsynaptic (clustered acetylcholine receptors or AChRs) and green-labeled nerve moieties (NF and SV2) were imaged on a PMT detector using a Leica TCS SP 8X, equipped with 488nm and 561nm wavelength lasers and appropriate filter combinations. The zoom factor is set to 1.28 times, and parameters such as laser intensity, confocal aperture and photomultiplier gain are kept constant among samples. For 488nm, 0.5% maximum laser power, gain was set at 740, offset was-1, and there was no average. For 561-nm, 2.6% maximum laser power, gain was set to 780, offset was-1, no average. Z-stacks were projected into a single image using LAS-X software (Leica), without using Z compensation. The full package micrographs were collected with a 10-fold objective lens and then stitched with LASX. Fine details were determined with a 63X/1.4NA oil immersion objective.

The area of the diaphragm surface and the area after the synaptesis were measured with Image J software. Statistical analysis GraphPad Prism software was used. Data were expressed as mean ± SEM using a two-tailed t-test. Statistically significant differences are shown as p <0.05, p <0.01 and p < 0.001.

Analysis of results

Figure 1 is a schematic illustration of the preparation of the diaphragm by muscle stretching; wherein Non-compressed represents an uncompressed control group and compressed represents a compressed experimental group; (A) is a schematic of the device used to press the diaphragm, (B) is a schematic of the diaphragm on plexiglas, when it is not pressed, (C) is a schematic of the diaphragm flattening and unfolding between the two plexiglas sheets after the device is closed, (D) is a schematic of the diaphragm remaining flat after the device is removed, (E) is a statistical plot of the length (ventral to dorsal) and width (widest distance in the semi-diaphragm) of the compressed and uncompressed diaphragms for 2.5 month old male mice, with n-3 mice per group, p < 0.01; (F) the area calculation method is characterized by comprising the following steps of (1) manually drawing a diaphragm outline in Image J software to obtain a statistical result graph of areas of stressed and unstressed diaphragms, wherein n is 3 mice per group, and p is less than 0.01; (G) is a graph of the statistical results of the thickness of the compressed and uncompressed diaphragms; to determine thickness, three points were measured around the central region of the diaphragm using background signal from one side of the sample until no signal was detected under confocal microscopy to obtain an average, n-3 mice, p < 0.001. (H) Is a comparison of muscle fiber stretch, actin filaments are labeled with Phalloidin-594, region H is between two bright actin bands, and the following 1, 2 are single muscle fiber images cut from the top, Phalloidin (red), DAPI, 4', 6-diamidino-2-phenylindole (blue).

As shown in fig. 1B and 1C, the compressed diaphragm expands uniformly, even after the device is removed, the diaphragm remains flat and expanded, as shown in fig. 1D. Both the length and width of the compressed diaphragm were increased, as shown in FIG. 1E, and showed about 2.5 times larger than the original area (compressed diaphragm area: 8.65. + -. 0.63 cm)²(ii) a Area of uncompressed diaphragm in control group: 3.49 +/-0.34 cm²，p<0.01, as shown in FIG. 1F). The thickness of the pressurized diaphragm was also reduced by 45% (thickness of uncompressed diaphragm: 141.6 + -3.6 μm; thickness of pressurized diaphragm: 78.2 + -0.7 μm, p)<0.001 as shown in fig. 1G).

Staining of actin with phaseolin-594 revealed an increase in space in region H, but no apparent stress injury (as shown in FIG. 1H). These data indicate that the method of the present invention can effectively stretch the diaphragm muscle while maintaining the integrity of the overall structure.

FIG. 2 is a schematic representation of NMJs in adult mouse diaphragms, where Non-compressed represents an uncompressed control group and compressed represents a compressed experimental group; (A) is a representative immunostaining image of an uncompressed adult diaphragm; the enlarged views of the areas in boxes 1-3 are arranged sequentially from left to right, with dense parallel green lines (the portions indicated by white arrows) being the autofluorescence signal emitted by the muscle fibers; NF/SV2 (green), BTX (red); (B) is a typical immunostained image of stressed adult diaphragm, with innervated NMJs shown in yellow, representing neurofilaments and SV2 overlaid on the endplates, with enlargements of the areas in boxes 1-3 arranged in left-to-right order, NF/SV2 (green), BTX (red), (C) is a statistic of the area post-synaptic of the unstressed and stressed diaphragms, with a total of 30 endplates measured per diaphragm, 3 mice per group, p <0.01, (D) is a fragmentation index statistic, 3 mice per group, a total of 30 endplates measured per diaphragm, 3 mice per group, insignificant, (E) is a representative image of acetylcholinesterase silver treated unstressed adult diaphragm, with enlargements of the areas in boxes 1-2 arranged in top-to-bottom order, (F) is a representative image of acetylcholinesterase silver treated stressed adult diaphragm, axons (dark brown); the enlarged views of the regions in the end plate (indigo), boxes 1-2 are arranged in sequence from top to bottom.

When the diaphragm is stretched by intrinsic forces only (i.e., PFA immobilized in the thoracic cavity), usually the immunolabeling occurs near the thinner dorsal/ventral region, or the area of injury usually due to facial resection (fig. 2A). Whereas bungarotoxins (-8 kDa) easily reach postsynaptic target surfaces (AChRs), antibodies (around 150kDa) hardly bind to antigens deep in the tissue, eventually leading to uneven staining of presynaptic organs (fig. 2A). The thickness of the sample affects the resolution of the image because of light scattering. However, the flatness after immunostaining (i.e., pressing the diaphragm after immunostaining) does not improve the image quality.

The entire innervation pattern (labeled with anti-NF and anti-SV 2) was clearly immunostained (as shown in fig. 2B, fig. 4 and 5). Innervated NMJs appear yellow, green (presynaptic terminals), red (post-synaptic AChRs), and a mixture of green and red. The size of the end plate is enlarged (non-pressed area: 314.70 + -19.90 μm²(ii) a Area under pressure: 429.10 +/-11.29 mu m²，p<0.01, fig. 2C), but no significant change in the fragmentation index, further supporting the preservation of structural integrity of the stressed NMJS (unstressed: 1.096 +/-0.013; pressing: 1.148 +/-0.047; fig. 2D).

The autofluorescence signal appeared to be less in the uncompressed diaphragm (figure 2A) compared to the compressed diaphragm (figure 2B). The background color of the compressed diaphragm is much lighter than that of the uncompressed diaphragm, probably because of less pigmentation in the fibers of the dilator muscles (see figures 2E and 2F). Thinner samples also allow more efficient transmission of light and easier viewing of the image. In summary, the pretreatment method of the present invention provides a convenient and versatile method to improve the visualization of innervation networks, not only for immunolabeling, but also for classical esterase silver staining.

Tissue transparency techniques like 3 DISCO/idsco, CUBIC are the most advanced techniques but require many time consuming steps before imaging (more than 10 days in total), image details do not improve when studying flat muscles like the diaphragm (Ert urk et al, 2012; Khan et al, 2016; Renier et al, 2014; Susaki et al, 2014; Williams et al, 2019), with the pre-treatment method of the present invention can be successfully completed within 2 days based on conventional immunostaining procedures. The pretreatment method of the present invention can help the penetration of the antibody without removing the fascia.

FIG. 3 shows SOD1^G93AMorphological changes in pathological NMJs in mice, where (A) is the end stage of disease (4 months old), SOD1^G93ARepresentative images of mouse diaphragm, a number of NMJs were denervated (portion indicated by white arrows), enlarged views of the regions in boxes 1-3 arranged sequentially from left to right, (B) age-matched control wild-type WT and SOD1^G93AThe method comprises the steps of (1) counting postsynaptic areas of mouse diaphragms, analyzing 90 end plates of each diaphragm, and analyzing 3 mice in each group without significance, (C) counting fragmentation indexes, analyzing 90 end plates of each diaphragm, and analyzing 3 mice in each group without significance, (D) counting nerve occupancy rate, analyzing more than 1000 end plates of each diaphragm, and analyzing 3 mice in each group, (E) high-power confocal images of NMJs, wherein WT represents high-power confocal images of NMJs of control wild type mouse diaphragms, and healthy NMJ images are displayed; 1-SOD1^G93ARepresents SOD1^G93AHigh power confocal images of NMJs of mouse diaphragm showing multiple innervated NMJs (white arrows); partially innervated NMJs (blue arrows); 2-SOD1^G93ARepresents SOD1^G93AHigh power confocal images of NMJs of mouse diaphragm, where completely denervated NMJs (purple arrows), NF/SV2 (green), BTX (red) are shown. SOD1^G93AThe mice represent mice of a model of progressive freezing disease (SOD1 eggs)Amino acid 93 of white mutated from glycine to alanine).

The invention shows SOD1^G93AGlobal staining of the diaphragm of the mouse ALS model, which enabled systematic assessment of morphological changes in NMJs (fig. 3A). SOD1 large in 4 months^G93AIn mice, there was no significant difference in postsynaptic area and fragmentation index (FIGS. 3B and 3C), but there were a large number of denervated and partially denervated NMJs (wild type WT: 4.02. + -. 1.66%; SOD 1) compared to wild type control group^G93A: 44.25 ± 5.67%, fig. 3D). The high power image shows detailed morphological features, healthy NMJs are characterized by the structural complexity of pre-and post-synaptic elements (fig. 3E above). Pathological features such as multiple innervation, partial innervation, and denervation were also clearly demonstrated (middle and bottom, fig. 3E), consistent with previous findings (Martin et al, 2015; Valdez et al, 2012). This indicates that the pretreatment method of the present invention enables effective analysis of the innervation of the diaphragm while preserving the different types of NMJ structural features.

It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Claims

1. A pretreatment method for integral immunostaining of an adult diaphragm is characterized by comprising the following steps:

(6) removing connective tissue from the surface of the diaphragm;

(9) the diaphragm was then washed with PBS buffer containing 0.1% glycine for 10 minutes at room temperature;

in the step (1) and the step (2), the flat plates are provided with at least one flat surface, through holes are arranged at four corners of each flat plate, and the through holes are symmetrically arranged;

in the step (1), the flat surface of the flat plate is contacted with an adult diaphragm;

in the step (1) and the step (2), the flat plate is made of transparent materials, and the thickness of the flat plate is larger than or equal to 6 mm.