Method for fixing, embedding and slicing crystalline lens

Abstract

The invention discloses a crystalline lens fixing, embedding and slicing method, which is characterized in that the hardness gradient of a crystalline lens is adjusted through two-step fixation (paraformaldehyde and acetic acid), and the stability of the slicing is enhanced by combining an outer support structure (pin and plastic soil), so that 8-10-micron high-quality slicing of large crystalline lenses (cattle, pigs and monkeys) is realized. Compared with a traditional method, the slice integrity rate is increased to 90% or above, the cell structure is clear, the method can be directly used for immunohistochemistry and fluorescence research, and the limitation that crystalline lens research depends on small animal models for a long time is solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological tissue sectioning, and particularly relates to a method for fixing, embedding and sectioning large lenses (such as bovine, porcine and monkey lenses), and is particularly suitable for immunohistochemistry or immunofluorescence staining research. Background Art

[0002] The lens is an important part of the eye's refractive system. It is a biconvex transparent tissue, fixed and suspended by zonular ligaments behind the iris and in front of the vitreous body. During the formation and development of the lens, newly differentiated lens fibers are located in the outer layer. As the fibers age, the old fibers are squeezed towards the center, dehydrated and hardened, thus forming an onion-like lens tissue, as Figure 1 shown. A large number of studies have shown that the protein content of the lens gradually decreases from the center to the periphery. The lenses of rats and mice have no accommodation ability. The lenses of cattle and pigs have an accommodation of about 2D, and the lenses of primates in their juvenile stage have an accommodation of about 10D - 15D.

[0003] The lens is the core tissue of the eye's refractive system. Its structure is dense, the water content gradually decreases from the center to the periphery, and the protein content is extremely high. In the prior art, tissue sectioning mainly uses the cryostat sectioning method and the paraffin sectioning method, but both have significant defects:

[0004] I. Cryostat sectioning method:

[0005] It is a method of quickly cooling the tissue to a certain hardness at low temperature and then sectioning. The steps are as follows:

[0006] 1. Specimen collection

[0007] (1) When collecting fresh tissue, it cannot come into contact with water. If the tissue itself has a lot of water, it can be blotted dry with filter paper (the purpose is to prevent phenomena such as ice crystal cavities from occurring during sectioning);

[0008] (2) The size of the collected tissue should be appropriate, and the thickness should be preferably 3 mm, and at most not exceed 5 mm (if the tissue is too large, the resistance during sectioning will increase, and knife marks and wrinkles are likely to occur).

[0009] 2. Embedding

[0010] (1) For the embedding machine, OTC or ordinary glue is generally used. The OTC embedding agent has a delicate texture, little damage to the blade, and is beneficial for sectioning;

[0011] (2) When embedding, the embedding agent should be evenly applied, and the embedding direction of the tissue should be determined. For example, lumens, skin, cyst walls, etc. should be vertically embedded;

[0012] (3) For tissues with a small volume, a little embedding agent can be first extruded to form a platform, and then the small tissue can be placed for embedding. AsFigure 2 as shown

[0013] 3. Freezing Temperature and Time

[0014] (1) Freezing temperature and time are crucial steps for frozen sections and should be adjusted according to the type and size of the tissue;

[0015] (2) If the freezing temperature is too low, the tissue will become too hard and the sections will break; if the temperature is too high, the tissue will not be hard enough and it will be difficult to section;

[0016] 4. Sectioning

[0017] (1) Sectioning can be started after the tissue and embedding medium have been frozen until they turn white;

[0018] (2) When sectioning, the right hand should turn the handwheel with uniform, gentle and slow force. The section thickness should be controlled at about 5 μm. The left hand holds a writing brush and gently presses on the edge of the tissue when the section is formed to prevent it from curling;

[0019] (3) The sections should be flattened and pasted on clean glass slides and immediately placed in the fixing solution for fixation. If the fixation is not timely, cell degeneration will occur and the cell nuclei in the sections will be blurred and foggy.

[0020] II. Paraffin Section Method:

[0021] Paraffin infiltration will further increase the hardness of the lens, especially in the central area, resulting in difficult sectioning;

[0022] The steps of dewaxing and rehydration may damage the morphology of lens cells and affect the staining effect.

[0023] In addition, existing studies mostly rely on small animal lenses, but they lack regulatory functions and have significant physiological differences from human eyes, resulting in great limitations in research. Therefore, there is an urgent need for a stable sectioning method for large lenses to promote lens pathology and immunology research.

[0024] The basic process of the paraffin section method is to first rinse the fixed tissue to be examined, dehydrate it (using ascending concentrations of ethanol, etc.), clear it (using xylene, etc.), infiltrate it with wax (using paraffin), then embed the tissue in a wax block with paraffin, and then section it with a microtome and stain it to make sections.

[0025] 1. Specimen Collection and Fixation

[0026] The small tissue specimens are first rinsed with 0.85% normal saline to wash away blood and stains. If it is tubular tissue, the contaminants in the lumen must be washed clean. The specimens should be fixed promptly after being taken. The purpose of fixation is to quickly allow the fixing solution to penetrate into the tissue interior and fix the tissue to maintain its original structure. Appropriate fixing solutions should be selected for different tissues. The ratio of the amount of fixing solution to the volume of the tissue block is generally 20:1.

[0027] 2. Rinsing

[0028] Before dehydration, the fixed tissue blocks must be rinsed with running water to remove the fixative. The tissue blocks should be wrapped with gauze and labeled, then placed in a metal mesh for rinsing with running water. Generally, tissues fixed with formaldehyde fixative are rinsed for 24 hours, and those fixed with Bouin's fixative are rinsed for 24 hours. Tissues fixed with AFA fixative do not require rinsing.

[0029] 3. Dehydration and Hardening

[0030] After fixation, the tissue is dehydrated after rinsing. The purpose is to replace the water in the tissue with alcohol or other solvents to harden the tissue, making it easier for oils or other clearing agents to penetrate. The tissue can be dehydrated using an automatic dehydrator or different concentrations of alcohol. Alcohol dehydration should be carried out step by step; skipping steps will cause strong shrinkage and deformation of the tissue blocks. The alcohol concentrations and process used for dehydration are as follows: 30%-50%-70%-80%-90%-95%-100%.

[0031] 4. Clearing

[0032] Clearing is a necessary step before sectioning by the paraffin section method. Its purpose is to replace absolute alcohol with a clearing agent such as mineral oil or vegetable oil, so that the melted paraffin can infiltrate into the tissue spaces. Therefore, the clearing agent must be soluble in both paraffin and alcohol. After being treated with the clearing agent, the tissue block appears transparent when observed against the light with the naked eye, so this process is called clearing.

[0033] Common clearing agents include xylene, benzene, toluene, etc. These are all clearing agents with strong penetration and are likely to make the tissue brittle. Therefore, the clearing time should be short, generally about 20 minutes.

[0034] 5. Impregnation

[0035] The impregnation process is to make the material used for embedding the tissue penetrate into all tissue spaces, and then solidify this material through various methods to embed the tissue material. Both the paraffin section method and the celloidin section method require the impregnation process. In the paraffin section method, heated paraffin penetrates into the tissue, while in the celloidin section method, celloidin penetrates into the tissue spaces. Before impregnation, the selected paraffin should be placed in containers and put in an incubator to precipitate its impurities. It is best to choose two types of paraffin, soft wax and hard wax. The melting point of soft wax is between 45-50°C, the melting point of hard wax is between 56-58°C, and hard wax at 60-62°C is rarely used.

[0036] The impregnation time of the tissue blocks in each wax cup is as follows:

[0037] The total time for waxing is generally as follows: for a tissue block of 0.5 cubic centimeters, the time is 6 - 8 hours; for a tissue block of 0.2 - 0.3 cubic centimeters, the time is 3 - 5 hours; for a tissue block of 0.1 - 0.2 cubic centimeters, the time is 2 - 3 hours. When dust or tissue fragments are found in the wax cup, let them settle or filter them before use. If the wax infiltration is incomplete, white opaque parts will appear after embedding, and at this time, wax infiltration needs to be carried out again.

[0038] 6. Embedding

[0039] Embedding means embedding the already infiltrated tissue block in the infiltrant. The paraffin embedding method is as follows:

[0040] At the end of wax infiltration, pour the paraffin with the same melting point as the fourth cup of paraffin, which has been preheated, into the folded wax trough. Then take out the tissue block from the fourth wax cup, place it in the wax trough and arrange it in position, paying attention to the placement direction of the tissue section. After placing the tissue, it can be cooled naturally or directly cooled in cold water.

[0041] 7. Plastic molding and sectioning

[0042] Fix the wax block on the microtome, adjust the distance and the required section thickness, and then turn the handle with the right hand to perform sectioning. Generally, the thickness of biological sections is 5 - 10 microns.

[0043] Do not pick up the cut sections by hand because body temperature can cause the wax sections to stick to the hand. The correct method is to pick them up with a brush or toothpick and then place them on a glass slide for flattening and sticking. As Figure 3 shown.

[0044] 8. Flattening and sticking the sections

[0045] The cut tissue wax sections often have wrinkles, so they must be flattened and stuck on the glass slide before staining, that is, stick the cut wax sections flat on the glass slide. The flattening temperature varies depending on the melting point of the paraffin. Generally, for paraffin sections with a melting point of 56 - 58°C, use warm water at 37°C for flattening; for paraffin sections with a melting point of 48 - 52°C, use warm water at 35°C for flattening. After the wax sections are fully flattened, take a clean glass slide, apply a thin layer of egg albumin glycerin on the glass slide, then pick up the wax sections in water. After picking up, straighten the wax sections, place them in a grid tray, dry them in an incubator at 38°C, and then they can be stained.

[0046] 9. Dewaxing, rehydration, and staining

[0047] Purpose of dewaxing: The dried sections need to be dewaxed and rehydrated before they can be stained in water-soluble staining solutions.

[0048] Purpose of rehydration: If the material after dewaxing is directly put into the staining agent without rehydration, the material will be severely deformed and even difficult to stain.

[0049] Purpose of staining: To make different structures in cell tissues show different colors and strong enough differences for easy observation.

[0050] Overall process: Xylene → 1 / 2 xylene + 1 / 2 absolute alcohol → 100% alcohol → 95% alcohol → 85% alcohol → Safranin (for more than 4 hours) → 95% alcohol → Fast Green (rapidly) → 95% alcohol (rapidly) → 100% alcohol → 1 / 2 xylene + 1 / 2 absolute alcohol → xylene → xylene. Each step above takes about 5 - 10 minutes, except for those with specified time.

[0051] The above are the existing two methods of tissue sectioning. Currently, almost all lens immunohistochemistry studies use the lenses of rats or mice. There are only a few literature reports on the staining of large lens sections, and most of them use frozen sections. Summary of the Invention

[0052] The existing methods of lens tissue sectioning have the following defects: [[ID=]13]

[0053] 1. Frozen sectioning requires that the tissue block must be small and be quickly frozen to prevent section rupture caused by inconsistent expansion rates in different parts of the tissue. However, the lens is a complete tissue wrapped by a capsule. After cutting, the cell arrangement will be disordered and deformed, and it can only be sectioned and fixed as a whole. There are differences in water content between the central and peripheral parts of bovine, porcine, and monkey lenses. During frozen sectioning, due to different expansion rates of the lens tissue, many cracks will appear in the lens sections, seriously affecting immunostaining.

[0054] 2. Paraffin sectioning will increase the hardness of the tissue. After the lens is fixed with formaldehyde, the hardness will increase, and it will further increase after paraffin infiltration. The center of the lens will become too hard to be sectioned.

[0055] 3. Currently, lens staining experiments can be carried out on small animals (rats and mice). However, due to the large differences in the eye structures and functions between these animals and human eyes, the lens completely lacks the ability to adjust. Limited by the research objects, many studies on the lens are in a stagnant state.

[0056] The present invention aims to provide a method for fixing, embedding, and sectioning lenses, which solves the problem of section fragmentation caused by uneven hardness and water content differences in large lenses, and avoids the damage to cell structures caused by paraffin penetration, so as to obtain complete and highly tough sections, which are suitable for immunohistochemistry and immunofluorescence staining.

[0057] Specifically, it includes the following steps:

[0058] (1) Fixing treatment: Immerse the lens successively in a 2% paraformaldehyde solution by mass concentration and a mixed solution of 2% paraformaldehyde and 5% acetic acid by mass concentration for two-step fixation;

[0059] (2) Slow drying: After the fixed lens is rinsed with PBS, it is placed in a sealed container and slowly dehydrated for 2-3 weeks;

[0060] (3) External support implantation: 4 pins are symmetrically inserted into the rear part of the lens, and the needle tips are wrapped with plasticine TPU (thermoplastic polyurethane) to form an external support structure; as Figures 4 - 6 shown;

[0061] (4) Paraffin embedding and sectioning: The lens with external support is directly embedded in paraffin, as Figure 7 and 8 shown; The section thickness is 8-10 microns;

[0062] (5) Staining application: The sections are directly immersed in the staining solution for immunohistochemical or immunofluorescent staining without a dewaxing step, as Figure 9 and 10 shown.

[0063] Preferably, in the two-step fixation: the fixation time with paraformaldehyde solution is 1-2 days, and the fixation time with the mixture of paraformaldehyde and acetic acid is 1-2 days.

[0064] Preferably, during the slow drying process, the inner wall of the container is wiped with water vapor every day, and the humidity is controlled at 85-95%.

[0065] Preferably, the length of the pins for the external support is 4-8 mm, the plasticine is thermoplastic polyurethane (TPU), and it is shaped after being heated to >70 °C.

[0066] Preferably, the paraffin embedding temperature is 60 °C, and the cooling time after embedding is >2 hours.

[0067] Preferably, after sectioning, it is unfolded with warm water at 40 °C, and the water bath time is 1-2 minutes.

[0068] Preferably, the lens is a bovine, porcine or simian lens with a diameter ≥7 mm.

[0069] Preferably, the insertion depth of the pins is 1 / 3-1 / 2 of the lens thickness and they are symmetrically distributed at 90°.

[0070] ]>Preferably, the contact area between the plasticine and the lens is 4-8 mm 2 , to provide uniform support force.

[0071] The present invention adopts the above technical solutions, and its advantages are that the method for fixing, embedding and sectioning the lens disclosed in the present invention is different from the frozen section and paraffin section introduced above. The present invention is made according to the characteristics of the lens tissue and is not applicable to other tissues.

[0072] First, the lens must have appropriate hardness during sectioning. Fixing biological tissues with formaldehyde can increase their hardness. However, due to the high content of lens proteins, after formaldehyde fixation, the central part of the lens will become too hard, making sectioning difficult. To solve this problem, the present invention adopts a two-step fixation method; fixing with 2% paraformaldehyde for 1 - 2 days can completely fix the peripheral part of the lens, with moderate hardness. Adding 5% acetic acid to 2% paraformaldehyde and then fixing for another 1 - 2 days, fixing biological tissues with acetic acid can reduce their hardness. At this time, the central part of the lens has moderate hardness under the combined fixation of formaldehyde and acetic acid, and the hardness of the entire lens tissue is evenly moderate.

[0073] Secondly, the characteristic that the water content of the lens gradually decreases from the center to the periphery makes it impossible to perform frozen sectioning. In paraffin sectioning, paraffin infiltration firmly fixes biological tissues in the paraffin block, but it will further increase the tissue hardness.

[0074] The present invention innovatively uses a new embedding and sectioning method, which can avoid the defects of the existing two methods. It can not only fix the lens tissue in the paraffin block but also does not require paraffin to infiltrate into cells. Insert four pins into the posterior part of the lens and wrap the pins with resin plasticine to increase the force-bearing area. These four pins inserted into the lens form an external support device. During sectioning, the external support device can fix the position of the lens without displacement. The spherical and smooth lens is embedded in the paraffin block. During sectioning, there is no force-bearing point and it is easy to rotate, but the lens with the added external support will not be displaced and can be sectioned without paraffin infiltration. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 It is a schematic diagram of the onion-like structure of the lens.

[0076] Figure 2 It is a schematic diagram of tissue embedding in the prior art.

[0077] Figure 3 It is a schematic diagram of paraffin sectioning in the prior art.

[0078] Figures 4 - 6 It is the orientation (such as symmetric distribution at the four corners) of inserting 4 pins into the posterior part of the lens in one embodiment of the present invention.

[0079] Figures 7 - 8 It is a schematic diagram of the paraffin embedding steps in one embodiment of the present invention.

[0080] Figures 9 - 10 It is a schematic diagram of the sectioning and picking-up steps in one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0081] The following further describes in detail the preferred embodiments of the present invention in conjunction with the accompanying drawings:

[0082] The following embodiments facilitate a better understanding of the present invention, but do not limit the present invention. The embodiments of this patent application are only examples and do not limit the protection scope of the present invention. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all purchased from conventional biochemical reagent companies unless otherwise specified.

[0083] Example 1: Porcine lens slice (diameter 10 mm)

[0084] Fixation:

[0085] Fix with a mixture of 2% paraformaldehyde and 5% acetic acid by mass concentration for 24 hours.

[0086] Drying:

[0087] Rinse with PBS 3 times, 5 minutes each time, place in a 50 ml blue-capped bottle, wipe the water vapor inside the bottle daily, keep the humidity inside the bottle at 90 - 95%, and dry for 2 weeks.

[0088] External support:

[0089] Insert 4 6-mm pins (depth 3 mm), heat the plasticine to 70 °C to wrap the pinheads, and the contact area is 4 mm 2 .

[0090] Embedding:

[0091] Embed in paraffin at 60 °C, and trim the wax block after cooling for 3 hours.

[0092] Sectioning:

[0093] The section thickness is 8 microns, unfold in warm water at 40 °C for 1 minute, use forceps to peel off the paraffin film around the lens section, and pick up the section with a glass slide.

[0094] Staining:

[0095] Directly perform Alexa Fluor 594 WGA immunofluorescence staining, observe with a fluorescence microscope, the cell structure is complete and the staining is uniform.

[0096] Example 2: Bovine lens slice (diameter 17.5 mm)

[0097] Fixation:

[0098] Fix with a mixture of 2% paraformaldehyde and 5% acetic acid by mass concentration for 48 hours.

[0099] Drying:

[0100] Dry for 3 weeks, and control the humidity at 85 - 90%.

[0101] External support:

[0102] Insert 4 8-mm pins (depth 4 mm), heat the plasticine to 70 °C and wrap the pinheads, contact area 8 mm 2 .

[0103] Embedding:

[0104] Embed in paraffin at 62 °C, trim the wax block after cooling for 4 hours.

[0105] Sectioning:

[0106] The section thickness is 10 μm, unfold in warm water at 40 °C for 1 minute, use forceps to peel off the paraffin film around the lens section, and pick up the section with a glass slide.

[0107] Staining:

[0108] Directly perform Alexa Fluor 594 WGA immunofluorescence staining. Observe with a fluorescence microscope, the integrity rate of the paraffin section is 95%, the lens cells are intact and there are no fragments (the fragmentation rate is reduced by 75% compared with traditional frozen sections).

[0109] Example 3: Monkey lens section (diameter 7 mm)

[0110] Fixation:

[0111] Fix with a mixture of 2% paraformaldehyde and 5% acetic acid and 5% acetic acid for 24 hours.

[0112] External support:

[0113] Insert the pin to a depth of 2.5 mm, heat the plasticine to 70 °C and wrap the pinhead, contact area 4 mm 2 .

[0114] Results:

[0115] The integrity rate of the paraffin section is 95%. After staining, the nuclei of the lens epithelium are clearly visible and the fiber layer is not broken.

[0116] The comparison data table between the examples of the present invention and the traditional method is shown in Table 1.

[0117] Table 1

[0118]

[0119] The lens sections are thin and have high toughness. The thickness of the lens sections cut by this method is 8-10 μm, which is much smaller than the thickness of frozen sections of 20-30 μm. When the crystal is stained, its toughness is very high and it is not easy to break, making the immunostaining easier to operate and facilitating large-scale immunohistochemical research.

[0120] The invention can successfully section and study large lenses such as cows, pigs, and monkeys, expanding the research objects in the field of lenses.

[0121] This method circumvents the disadvantages of traditional paraffin sectioning and frozen sectioning. According to the tissue characteristics of the lens, a new method of fixation, embedding and sectioning is designed. By adding external support to the lens, the disadvantage of the easily displaced round and smooth lens is overcome.

[0122] The above content is a further detailed description of the present invention in combination with specific preferred embodiments. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention pertains, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, which should all be regarded as belonging to the protection scope of the present invention.

Claims

1. A method for fixing, embedding and sectioning a lens, characterized in that It includes the following steps: (1) Fixation treatment: Immerse the lens in a paraformaldehyde solution with a mass concentration of 1.8% - 2.1% and then in a mixed solution of 1.8% - 2.1% paraformaldehyde and 2% - 5% acetic acid successively for two-step fixation; (2) Slow drying: After the fixed lens is rinsed with PBS, place it in a closed container and slowly dehydrate for 2 - 3 weeks; (3) External support implantation: Insert 4 pins symmetrically at the rear of the lens, and wrap the needle tips with plasticine to form an external support structure; (4) Paraffin embedding and sectioning: Embed the lens with external support directly in paraffin, and the section thickness is 8 - 10 microns; (5) Staining application: Immerse the sections directly in the staining solution for immunohistochemistry or immunofluorescence staining without a dewaxing step.

2. The method according to claim 1, characterized in that, In the two-step fixation: The fixation time with paraformaldehyde is 1 - 2 days, and the fixation time with acetic acid is 1 - 2 days.

3. The method according to claim 1, wherein During the slow drying process, wipe the water vapor on the inner wall of the container every day, and control the humidity at 85% - 95%.

4. The method according to claim 1, wherein The length of the pins for the external support is 5 - 8 mm, and the plasticine is thermoplastic polyurethane, which is shaped after being heated to >70°C.

5. The method according to claim 1, characterized in that, The paraffin embedding temperature is 60°C, and the cooling time after embedding is >2 hours.

6. The method according to claim 1, wherein After sectioning, it is unfolded with warm water at 40°C, and the water bath time is 1 - 2 minutes.

7. The method according to claim 1, characterized in that, The lens is a bovine, porcine or monkey lens with a diameter ≥7 mm.

8. The method according to claim 4, wherein The insertion depth of the pins is 1 / 3 - 1 / 2 of the lens thickness, and they are symmetrically distributed at 90°.

9. The method according to claim 1, characterized in that, The contact area between the plasticine and the lens is 4 - 8 mm² to provide uniform support force.

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