Rapid sheet laying method for cochlea of large animal

Abstract

The invention belongs to the field of ear science and biomedical research, and particularly relates to a rapid sheet laying method for a cochlea of a large animal, which comprises the following steps: taking a material from the cochlea of the large animal, fixing, directly cutting and dissecting, and then decalcifying, trimming and flaking. According to the method, the cochlea spreading and flaking time of the large animal is shortened from 4 weeks to 4 days the same as that of a small animal, and rapid observation of cochlea tissues of large animals such as pigs and the like can be realized.

Description

Technical Field

[0001] The invention belongs to the field of otology and biomedical research, and in particular relates to a rapid slab-laying method based on freshly collected large animal cochlear specimens. Background Art

[0002] Cochlear flat mount is a morphological research method that uses a fluorescence microscope to visually and accurately image and locate tissue derived from the cochlea, based on the principle of specific binding of fluorescein to target antigens.

[0003] The steps of conventional cochlear flat-slicing are: sampling, fixation, decalcification, dissection, and preparation (sealed punching, primary antibody incubation, rinsing, secondary antibody incubation, rinsing, DAPI staining and sealing). Obtaining tissues such as the basilar membrane and spiral ligament of the cochlea is a key step in cochlear flat-slicing. Because the cochlea of ​​experimental animals is wrapped in a hard bone shell, forced dissection is likely to damage the cochlear tissue to be observed, and further processing is routinely required after decalcification. Due to differences in the species and age of experimental animals, the decalcification time varies greatly. The decalcification time of the cochlea of ​​mice, guinea pigs, and pigs is approximately 10 hours, 2 days, and 3-4 weeks. Therefore, from sampling to the completion of cochlear flat-slicing, the time taken for mouse, guinea pig, and pig specimens is approximately 4 days, 6 days, and 4 weeks. The preparation time of pig cochlear flat-slicing is much longer than that of rodents, which greatly prolongs the research cycle.

[0004] Existing studies have shown that miniature pigs have great potential as large animal models in otology research and are an important tool in otology translational medicine research. In translational medicine research, some experiments have strict requirements on specimen processing time, such as tracing studies of miniature pigs introduced through the inner ear. Because the tracing dyes or fluorescent markers introduced into the cochlea may have fluorescence quenching over time, it is necessary to complete the observation of the cochlear tissue of the experimental animal in a relatively short time.

[0005] Existing technology is unable to solve the technical difficulties of rapid cochlear spreading in large animals such as pigs, which has become a huge obstacle to this type of research. Summary of the invention

[0006] The purpose of the present invention is to overcome the defects of the prior art and propose a rapid cochlear slicing method for large animals such as pigs. The present invention shortens the preparation time of cochlear slicing of large animals from 4 weeks to 4 days, which is the same as that of small animals, and can realize the rapid observation of cochlear tissue of large animals such as pigs.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] A rapid slab-laying method for the cochlea of ​​a large animal, the rapid slab-laying method comprising:

[0009] After the cochlea of ​​large animals is collected and fixed, it is directly cut and dissected, and then decalcified, trimmed and sliced.

[0010] The method for obtaining the materials is as follows: after a large animal is euthanized, the cochlea is taken out.

[0011] The fixation is as follows: the removed cochlea is moved under a stereo microscope, a hole is drilled from the cochlear apex, the remaining ossicles are removed, the stapes is punctured, the vestibular window is opened, the round window membrane is torn off, and 4% paraformaldehyde fixative is slowly perfused from the hole drilled at the cochlear apex, and this is repeated for more than 3 times; the cochlea is placed in a centrifuge tube filled with 4% paraformaldehyde fixative, and placed in a 4°C refrigerator overnight.

[0012] The cutting is as follows: a fixed cochlear specimen is taken, and a plane perpendicular to the modiolus is imagined through a line from the cochlear apex to the oval window to divide the cochlea into two; a left half and a right half of the cochlea are obtained, wherein one half of the cochlea contains cochlear tissues of the middle turn and the bottom turn, and the other half of the cochlea contains cochlear tissues of the top, middle and bottom turn.

[0013] The dissection is as follows: 1) Free cochlear transit tissue:

[0014] Take half of the cochlear tissue containing the apical, middle and basal turns, find the cochlear membranous labyrinth middle turn and its upper and lower boundaries under a low-power microscope, cut the modiolus outward along the junction of the middle and basal turns of the cochlea, separate the middle turn from the basal turn, cut the modiolus along the junction of the apical turn and the middle turn, separate the middle turn from the apical turn;

[0015] 2) Obtaining cochlear transit tissue:

[0016] Carpet-type separation was performed along the inner wall of the cochlear transit bone shell to obtain the cochlear transit tissue;

[0017] 3) Obtaining cochlear apex tissue:

[0018] Find the upper and lower limits of the apical turn of the cochlear membranous labyrinth. The upper limit of the apical turn is the cochlear apex. A hole has been drilled during cochlear perfusion and fixation, and the soft tissue of the cochlear apex is basically separated from the bone shell. Carefully separate the inner wall of the cochlear apical turn bone shell in a carpet-like manner to obtain the cochlear apical turn tissue.

[0019] 4) Obtaining cochlear basal tissue:

[0020] Cut the cochlear spiral plate, cut the spiral ligament and the basement membrane, separate the spiral plate from the cochlear shell, and remove the hook part of the cochlear soft tissue; then refer to steps 1) and 2) to obtain the remaining cochlear basal turn tissue;

[0021] 5) Take half of the cochlear tissue including the middle turn and the basal turn, and separate the soft tissues of the middle turn and the basal turn according to steps 1) and 2).

[0022] The decalcification is as follows: the obtained cochlear soft tissue specimens of the apical turn, the middle turn and the basal turn are placed in a decalcification solution in groups and placed for decalcification at room temperature for 4 hours.

[0023] The trimming is as follows: under a high-power stereo microscope, the apical, middle and basal cochlear tissues are distinguished, the vestibular membrane and the tectorial membrane are removed, and the vascular striae, spiral ligaments, bony spiral plates, cochlear nerves and basilar membranes are dissected one by one in the order of dissection to obtain a plurality of spread specimens.

[0024] The preparation includes blocking and punching, primary antibody incubation, rinsing, secondary antibody incubation, rinsing, staining and sealing.

[0025] Preferably, the large animal is a miniature pig, rabbit, dog or monkey.

[0026] According to a preferred embodiment of the present invention, a rapid slicing method for pig cochlea is provided. After a miniature pig is euthanized, the cochlea is sampled and fixed, and then directly cut and dissected, and then decalcified and sliced.

[0027] The method specifically comprises:

[0028] 1. Sample collection: Euthanize the pig and remove the cochlea.

[0029] 2. Fixation. Quickly move the freshly harvested cochlea to a stereo microscope, use an 18G syringe needle to drill a hole from the cochlear apex, remove the remaining ossicles, puncture the stapes, open the vestibular window, and continue to tear off the round window membrane. Use a 1ml pipette tip to fill it with 4% paraformaldehyde fixative, and slowly perfuse it from the hole drilled at the cochlear apex, repeating more than 3 times. Place the cochlea in a centrifuge tube filled with 4% paraformaldehyde fixative and place it in a 4°C refrigerator overnight.

[0030] 3. Cut the cochlea. Take the fixed pig cochlear specimen, and imagine a plane perpendicular to the modiolus through the line connecting the cochlear apex to the oval window. Use a micro-adjustable electric grinder to cut the cochlea in two at a time at a speed of 13000-16000r / min. Obtain a left and right half of the cochlea, one half of the cochlea contains the cochlear tissue of the middle and bottom turns, and the other half of the cochlea contains the cochlear tissue of the top, middle and bottom turns. The cutting edges of the two halves of the cochlea are smooth, and the internal structure is clear and continuous. During the cutting process, excessive tissue loss caused by back and forth cutting should be avoided. At the same time, the cutting site should be cooled with slow-flowing normal-temperature running water to prevent the cutting site from being too hot and changing the physical and chemical properties of the tissue.

[0031] 4. Peel off the basilar membrane, spiral ligament, cochlear nerve and other inner ear tissues.

[0032] (1) Free the cochlear transit tissue. Take half a piece of cochlear tissue containing the apical, middle, and basal turns. Under a low-power microscope, find the cochlear membranous labyrinth transit and its upper and lower boundaries. Use an 18G syringe needle to cut the modiolus outward along the junction of the cochlear middle and basal turns to separate the transit from the basal turn. Cut the modiolus along the junction of the apical and middle turns to separate the transit from the apical turn.

[0033] (2) Obtaining the cochlear transit tissue. Bend the tip of the 18G syringe needle at 135° along the oblique side, with a bending length of about 2 mm. Use the bent 18G syringe needle to carefully separate the inner wall of the cochlear transit bone shell in a carpet-like manner to obtain the cochlear transit tissue.

[0034] (3) Obtain the cochlear apical turn tissue. Find the upper and lower boundaries of the cochlear membranous labyrinth apical turn. The upper boundary of the apical turn is the cochlear apex, which has been drilled during cochlear perfusion and fixation, and the cochlear apex soft tissue is basically separated from the bone shell. Use a bent 18G syringe needle to carefully separate and obtain the cochlear apical turn tissue along the inner wall of the cochlear apical turn bone shell.

[0035] (4) Obtain the cochlear basal turn tissue. The structure of the starting section of the cochlear basal turn near the round window (the hook of the basilar membrane) is quite different from the rest. When separating this part, use an 18G syringe needle to cut off the cochlear bony spiral plate, and use microscissors to cut the spiral ligament and basilar membrane. Continue to separate the bony spiral plate from the cochlear bony shell. Use a bent 18G syringe needle to carefully separate until this piece of hooked cochlear soft tissue is removed. Then refer to the methods described in steps (1) and (2) to obtain the remaining cochlear basal turn tissue.

[0036] (5) For the other half of the cochlea, refer to steps (1) and (2) to separate the middle and bottom soft tissues.

[0037] 5. Decalcify at room temperature for 4 hours. Place the cochlear soft tissue specimens obtained from the top, middle and bottom turns in EP tubes filled with decalcification solution (10% EDTA, pH 7.4) and decalcify at room temperature to reduce the hardness of the bone spiral plate and make the paving smoother. After about 4 hours, no obvious uniform fine granular bone residue is found under a low-power microscope, and then proceed to the next step.

[0038] 6. Trim the specimen. Under a high-power stereomicroscope, distinguish the top, middle, and bottom cochlear tissues, remove the vestibular membrane and tectorial membrane with gossamer forceps, and then use microscissors, gossamer forceps, 15# stainless steel surgical blades and other tools to dissect the vascular striae, spiral ligaments, bony spiral plates, cochlear nerves, and basilar membranes one by one to obtain multiple spread specimens.

[0039] 7. Immunofluorescence staining: Complete the immunofluorescence staining and slide preparation according to the steps of blocking the holes, incubating the primary antibody at 4°C overnight, rinsing, incubating the secondary antibody, rinsing, spreading the slides and sealing the slides.

[0040] 8. Observe with laser confocal microscopy.

[0041] The present invention proposes a method for rapid cochlear slicing of large animals such as pigs, which can skip the long (about 4 weeks) decalcification process of large animal cochlear specimens and realize rapid cochlear slicing specimen preparation, which takes basically the same time as that of small animals such as mice. Using the method of the present invention, cochlear slicing observation of large animals such as pigs can be realized, and the observation effect is similar to that of conventional processing methods. Using the method of the present invention, it can be used for the observation of cochlear specimens of large animals with strict observation time requirements.

[0042] The method of the present invention directly cuts the cochlea without decalcification after the cochlear specimen is fixed. The "one-size-fits-all" cutting method along the central axis can reduce the loss of tissue specimens. The selected cutting position can fully expose the internal structure of the cochlea and obtain relatively complete tissue specimens. The cochlear soft tissue is carefully dissected and dissected from the cut cochlea, and then after a short-term decalcification treatment (about 4 hours), the basilar membrane, spiral ligament, vascular striae, bony spiral plate, cochlear nerve and other tissues of the top, middle and bottom turns can be quickly obtained.

[0043] The method of the present invention can break through the time limit for processing cochlear specimens of large animals such as pigs, shorten the experimental cycle, and improve research efficiency; it can solve the problem of observing easily quenched tracer dyes or fluorescent markers in the inner ear of large animals; the method is easy to operate and can be promoted and applied.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The method of the present invention is easy to operate, can maintain the morphology of cochlear tissue intact, greatly shortens the specimen processing time, and can realize the rapid observation of cochlear spreads of large animals such as pigs. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 It is the main tool and pig cochlear specimen used in the present invention;

[0047] Figure 2 This is a schematic diagram of cutting the pig cochlea, and the plane indicated by the black line is the cutting plane;

[0048] Figure 3 It is a schematic diagram of the processing of some tissues of the cochlea after cutting; the four points a, b, c, and d marked along the black line are located in the cochlear transit, and the line segments ab and cd are the places where the modiolus is severed when obtaining the cochlear transit tissue; the four points e, f, g, and g marked along the black line are located in the cochlear basal turn; the line segments ef, fg, gh, and he are the starting segments of the separation of the cochlear basal turn respectively;

[0049] Figure 4 is the harvested cochlear tissue;

[0050] Figure 5 It is the trimmed cochlear tissue;

[0051] Figure 6This is an immunofluorescence staining image of the cochlea; the cochlear basilar membrane, spiral lamina, cochlear nerve, vascular stria, and spiral ligament obtained by the cochlear rapid spreading method were subjected to immunofluorescence staining, and the magnification was 20 times; blue is the cell nucleus labeled with DAPI, green is the nerve fibers or ganglion cells labeled with Rabbit anti-βⅢtubulin, and red is the hair cells and skeleton labeled with Phalloidin. DETAILED DESCRIPTION

[0052] The technical solution of the present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0053] Embodiment 1:

[0054] A method for rapid sizing of porcine cochlea.

[0055] Cochlear swabbing process:

[0056] Sampling, fixation, cutting, dissection, decalcification, specimen trimming, and slide preparation (blocking and punching, primary antibody incubation, rinsing, secondary antibody incubation, rinsing, DAPI staining and sealing).

[0057] The tool used for rapid paving of the present invention is as follows Figure 1 As shown:

[0058] Figure 1 In the figure, a is an 18G syringe needle, b is a membranous labyrinth stripper, c is a microscissors, d is a hairpin forceps 1, e is a hairpin forceps 2, f is a 15# stainless steel surgical blade and handle, g is a pig cochlear specimen in a tube, and h is a micro adjustable electric grinder.

[0059] Among them, the b-membranous labyrinth stripper is modified from an 18G syringe needle. The specific modification method is: the tip of the 18G syringe needle is bent 135° along the oblique side, with a bending length of about 2mm, and the sharp edge of the needle is polished with sandpaper to become blunt to avoid puncturing or scratching structures such as the spiral ligament.

[0060] Yunnan small-eared pigs of about 4 months old and weighing 18.5kg-19.8kg were used as the source of the experimental cochleae. The PBS solution used in the embodiment had a freshly prepared concentration of 0.01 mol / L and a pH of 7.4.

[0061] The specific steps are as follows:

[0062] 1. Sample collection. The miniature pigs used in the experiment were euthanized, and their heads were split in half from the center of the skull after decapitation. The brain tissue was opened, the dura mater was torn off, and the cochlea was slowly pried out with the tip of surgical scissors until it was removed.

[0063] 2. Fixation. Quickly move the freshly harvested cochlea to a stereo microscope, use an 18G syringe needle to drill a hole from the cochlear apex, remove the remaining ossicles, puncture the stapes, open the vestibular window, and continue to tear off the round window membrane. Use a 1ml pipette tip to fill it with 4% paraformaldehyde fixative, and slowly perfuse it from the hole drilled at the cochlear apex, repeating more than 3 times. Place the cochlea in a centrifuge tube filled with 4% paraformaldehyde fixative and place it in a 4°C refrigerator overnight.

[0064] 3. Cut the cochlea.

[0065] Take the fixed pig cochlear specimen and imagine a plane perpendicular to the modiolus along the line from the cochlear apex to the oval window (see Figure 2 ), using a micro adjustable electric grinder (see Figure 1 ), the cochlea was split into two at a speed of 13000-16000r / min. One left and one right half of the cochlea were obtained, one half of the cochlea contained the cochlear tissue of the middle and bottom turns, and the other half of the cochlea contained the cochlear tissue of the top, middle and bottom turns (see Figure 3 ). The cutting edges of the two halves of the cochlea are smooth, and the internal structure is clear and continuous. During the cutting process, it is necessary to prevent excessive tissue loss caused by back and forth cutting. At the same time, the cutting area should be cooled with slow-flowing normal-temperature running water to prevent the cutting area from being too hot and changing the physical and chemical properties of the tissue.

[0066] 4. Peel off the basilar membrane, spiral ligament, cochlear nerve and other inner ear tissues.

[0067] (1) Free the cochlear transit tissue. Take half of the cochlear tissue containing the apical, middle, and basal turns. Under a low-power microscope, find the cochlear membranous labyrinth transit and its upper and lower boundaries. Insert an 18G syringe needle along the junction of the cochlear middle and basal turns ( Figure 3 Cut the modiolus outward along the middle ab line, separate the middle and bottom turns, and follow the junction of the top and middle turns ( Figure 3 Cut off the modiolus (in the direction of the middle CD connection line) to separate the middle rotation and the top rotation.

[0068] (2) Obtaining the cochlear transit tissue. Bend the tip of the 18G syringe needle at 135° along the oblique side, with a bending length of about 2 mm. Use the bent 18G syringe needle to carefully separate the inner wall of the cochlear transit bone shell in a carpet-like manner to obtain the cochlear transit tissue.

[0069] (3) Obtain the cochlear apical turn tissue. Find the upper and lower boundaries of the cochlear membranous labyrinth apical turn. The upper boundary of the apical turn is the cochlear apex, which has been drilled during cochlear perfusion and fixation, and the cochlear apex soft tissue is basically separated from the bone shell. Use a bent 18G syringe needle to carefully separate and obtain the cochlear apical turn tissue along the inner wall of the cochlear apical turn bone shell.

[0070] (4) Obtain the cochlear basal turn tissue. The structure of the initial segment of the cochlear basal turn near the round window (the hook of the basilar membrane) is quite different from the rest of the cochlear basal turn. When separating this part, you need to hold an 18G syringe needle from e to f (see Figure 3 ) Cut off the spiral plate of the cochlear bone, and use microscissors to cut the spiral ligament and basement membrane along the direction from e to f. Continue from f to g (see Figure 3 ) Separate the spiral plate from the cochlear shell. Use a bent 18G syringe needle from g to h (see Figure 3 ) Carefully separate until the soft tissue of the hook part of the cochlea is removed. Then, refer to the method described in steps (1) and (2) to obtain the remaining cochlear basal tissue.

[0071] (5) For the other half of the cochlea, refer to steps (1) and (2) to separate the middle and bottom soft tissues.

[0072] 5. Decalcify at room temperature for 4 hours. Figure 4 ) were grouped and placed in EP tubes filled with decalcification solution (10% EDTA, pH 7.4) and placed at room temperature for decalcification to reduce the hardness of the bone spiral plate and make the slab smoother. After about 4 hours, no obvious uniform fine granular bone residue was found under a low-power microscope, and the next step could be continued.

[0073] 6. Trim the specimen. Under a high-power stereomicroscope, distinguish the apical, middle, and basal cochlear tissues, remove the vestibular membrane and tectorial membrane with gossamer forceps, and then use microscissors, gossamer forceps, 15# stainless steel surgical blades and other tools to dissect the vascular striae, spiral ligaments, bony spiral plates, cochlear nerves, and basilar membranes one by one to obtain multiple spread specimens (see Figure 5 ).

[0074] 7. Immunofluorescence staining.

[0075] (1) Block the perforations. Add 100ul of 10% goat serum in PBST solution to the samples in the above groups. Place at room temperature for 2 hours.

[0076] (2) Primary antibody incubation. Mix the primary antibody Rabbit anti-βⅢtubulin at 1:400 in PBST solution and shake well. Add 100ul of the prepared primary antibody to each of the above groups. Before adding the primary antibody, remove the blocking solution. After the operation is completed, place the samples in a dark box and store them in a refrigerator at 4℃ overnight.

[0077] (3) Rinse. The next day, remove the sample and remove the primary antibody. Rinse with PBS solution three times, 5 min each time.

[0078] (4) Secondary antibody incubation. Dissolve the secondary antibody Alexa Fluor 488goat-anti Rabbit IgG 1:500 and Phalloidin 1:1000 in PBST solution and shake well. Add 100ul of the prepared secondary antibody to the above groups in turn. Before adding the secondary antibody, remove the PBS solution. Keep away from light during the whole process. Finally, put the samples in a dark box and incubate at room temperature for one hour.

[0079] (5) Rinse. Remove the secondary antibody by aspiration. Rinse with PBS solution three times, 5 min each time.

[0080] (6) Spreading and sealing. Add 1-2 drops of DAPI on the slide, gently place the specimen in DAPI and immerse it, making sure that the basement membrane faces upward and other tissues are spread out flat. After DAPI staining for about 3 minutes, cover with a coverslip to ensure that there are no obvious bubbles, and seal the slide with neutral gum. The slide preparation is complete.

[0081] 8. Laser confocal microscopy observation, the results are as follows Figure 6 The results showed that the observation of various structures of the cochlea was comprehensive.

[0082] The present invention is applied to the cochlear slabs of large animals such as pigs that are wrapped in hard bone shells. After fixation, the specimens do not need to be decalcified, saving about 4 weeks of specimen processing time. A method of obtaining the soft tissue inside the cochlea by cutting and peeling is adopted: the cochlear bone shell is opened by cutting with a handheld metal saw, and after the internal structure of the cochlea is fully exposed, the soft tissue inside the cochlea is peeled and obtained.

[0083] The present invention first cuts the fixed cochlear specimen, peels off the tissue inside the cochlea and then decalcifies it. Through the innovative adjustment of the cochlear slicing process, the time required for specimen processing is greatly shortened.

[0084] In order to meet the needs of specimen processing, the present invention manufactures a membranous labyrinth stripper which is convenient for carpet-like separation of the spiral ligament of the cochlea from the bone shell, and can avoid damage to inner ear tissues such as the spiral ligament and the basilar membrane while effectively and quickly performing the stripping.

[0085] The process parameters (such as temperature, time, etc.) of the present invention can realize the method by taking upper and lower limits and interval values, and the embodiments are not listed one by one here.

[0086] Any contents not described in detail in the present invention can be based on the conventional technical knowledge in the art.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention is described in detail with reference to the embodiments, it should be understood by those skilled in the art that any modification or equivalent replacement of the technical solutions of the present invention does not depart from the spirit and scope of the technical solutions of the present invention and should be included in the scope of the claims of the present invention.

Claims

1. A rapid slab-laying method for the cochlea of ​​a large animal, the rapid slab-laying method comprising: After the cochlea of ​​large animals is collected and fixed, it is directly cut and dissected, and then decalcified, trimmed and sliced.

2. The rapid paving method according to claim 1, characterized in that: The materials are: After euthanasia of the animal, the cochlea was removed.

3. The rapid paving method according to claim 1, characterized in that: The fixing is: Move the removed cochlea under a stereomicroscope, drill a hole from the cochlear apex, remove the remaining ossicles, puncture the stapes, open the vestibular window, tear off the round window membrane, and slowly infuse 4% paraformaldehyde fixative from the hole drilled at the cochlear apex. Repeat this for more than 3 times. Place the cochlea in a centrifuge tube filled with 4% paraformaldehyde fixative and put it in a 4°C refrigerator overnight.

4. The rapid paving method according to claim 1, characterized in that: The cutting is: Take the fixed cochlear specimen, and divide the cochlea into two by imagining a plane perpendicular to the modiolus through the line connecting the cochlear apex to the oval window; obtain a left and a right half of the cochlea, one half of the cochlea contains the cochlear tissue of the middle and basal turns, and the other half of the cochlea contains the cochlear tissue of the apical, middle and basal turns.

5. The rapid paving method according to claim 1, characterized in that: The anatomy is: 1) Free cochlear transit tissue: Take half of the cochlear tissue containing the apical, middle and basal turns, find the cochlear membranous labyrinth middle turn and its upper and lower boundaries under a low-power microscope, cut the modiolus outward along the junction of the middle and basal turns of the cochlea, separate the middle turn from the basal turn, cut the modiolus along the junction of the apical turn and the middle turn, separate the middle turn from the apical turn; 2) Obtaining cochlear transit tissue: Carpet-type separation was performed along the inner wall of the cochlear transit bone shell to obtain the cochlear transit tissue; 3) Obtaining cochlear apex tissue: Find the upper and lower limits of the apical turn of the cochlear membranous labyrinth. The upper limit of the apical turn is the cochlear apex. A hole has been drilled during cochlear perfusion and fixation, and the soft tissue of the cochlear apex is basically separated from the bone shell. Carefully separate the inner wall of the cochlear apical turn bone shell in a carpet-like manner to obtain the cochlear apical turn tissue. 4) Obtaining cochlear basal tissue: Cut the cochlear spiral plate, cut the spiral ligament and the basement membrane, separate the spiral plate from the cochlear shell, and remove the hook part of the cochlear soft tissue; then refer to steps 1) and 2) to obtain the remaining cochlear basal turn tissue; 5) Take half of the cochlear tissue including the middle turn and the basal turn, and separate the soft tissues of the middle turn and the basal turn according to steps 1) and 2).

6. The rapid paving method according to claim 1, characterized in that: The decalcification is: The cochlear soft tissue specimens obtained from the apical, middle and basal turns were grouped and placed in decalcification solution and left to decalcify for 4 hours at room temperature.

7. The rapid paving method according to claim 1, characterized in that: The pruning is: Under a high-power stereomicroscope, the apical, middle, and basal cochlear tissues were distinguished, the vestibular membrane and tectorial membrane were removed, and the vascular stria, spiral ligament, bony spiral plate, cochlear nerve, and basilar membrane were dissected one by one in the order of dissection to obtain multiple spread specimens.

8. The rapid paving method according to claim 1, characterized in that: The preparation includes blocking and punching, primary antibody incubation, rinsing, secondary antibody incubation, rinsing, staining and sealing.

9. The rapid paving method according to claim 1, characterized in that: The large animal is a small pig, a rabbit, a dog or a monkey.

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