Manufacturing method of slices of various tissues and organs of rat

Abstract

The invention relates to a manufacturing method of tissue and organ slices of a rat. The preparation method mainly comprises the following steps: before slicing, pre-cooling a wax block, adding a room-temperature cold bath during slicing, and dropwise adding absolute ethyl alcohol into pure water. According to the method provided by the invention, the sheet unfolding effect can be softer, flatter and free of bubbles. The effect is more flat, and the number of wrinkled slices is small.

Description

Technical Field

[0001] The present application relates to the field of life science technology, and specifically to a method for preparing slices of various tissues and organs of rats. Background Art

[0002] With the continuous development of pathological detection technology, its application scope is becoming more and more extensive, such as rapid clinical diagnosis, scale-up of animal disease models, standardized evaluation, etc. Therefore, the quality of animal tissue slices has also attracted the attention of pathological technicians.

[0003] Rats have gradually become an important model animal for studying various human diseases due to their large size, easy handling, and obvious pathological characteristics.

[0004] The particularity of rat tissue, such as the soft texture of rat liver, loose lung structure, and hair contamination, is different from that of human tissue preparation. When making rat tissue slices, there are limitations such as easy breakage, uneven thickness, and hair adhesion. These technical limitations directly lead to poor preparation and even the need for re-slicing to a large extent.

[0005] Therefore, we need to develop a simple, efficient, high-quality slide spreading and retrieval technology that is worthy of promotion in various laboratories and hospital pathology departments, so as to improve the quality of pathology slide preparation and lay a solid foundation for animal pathology diagnosis. Summary of the invention

[0006] In order to solve or partially solve the problems existing in the related art, the present application provides a method for preparing slices of various tissues and organs of rats, which can make the slices flat and reduce wrinkles.

[0007] The present application provides a method for preparing slices of various tissues and organs of rats, the method comprising the following steps:

[0008] 1) Prepare water for spreading the slices and put the slices in a cold water bath; the temperature of the cold water bath is room temperature;

[0009] 2) Place the animal tissue wax block in pure water containing anhydrous ethanol, and then store it at 18-24°C for later use;

[0010] 3) placing the pre-cooled wax block of the animal tissue in 2) on a microtome for slicing; the initial trimming thickness of the microtome is set to 20-30um; the fine trimming thickness of the microtome is set to 2-10um;

[0011] 4) screening the slices in 3), and placing the screened slices into a glass jar and a slice bleaching instrument for bleaching respectively;

[0012] 5) baking the slices after blanching in 4) on a slice drying machine; the baking time is 10-15 minutes; the baking temperature is 60-61°C.

[0013] Furthermore, the amount of anhydrous ethanol in the pure water in 2) is as follows: 2-3 drops of anhydrous ethanol are added to 500 mL of pure water.

[0014] Furthermore, the water bath temperature of the slide bleaching apparatus is 40-41°C.

[0015] Furthermore, the specific operation steps for slicing in 3) are: pull the wax block loading rod forward with one hand, hold the wax block with one hand and then release the loading rod to clamp the loaded animal tissue wax block; adjust the position of the animal tissue wax block to a position that the blade can cut; when slicing, hold the brush in the left hand to guide the slice to stretch it.

[0016] Beneficial Effects

[0017] 1. The method for preparing slices of rat tissues and organs provided in the present application is to add anhydrous ethanol to pure water at room temperature for pre-slicing, and then enter the slice spreading machine in a hot water tank (the temperature is set at 40-41°C), increase the water temperature gradient in the slice spreading process, and then optimize the baking time and temperature to make the sliced ​​tissue more stretched and reduce wrinkles, folding and peeling.

[0018] 2. The method for preparing slices of rat tissues and organs provided in the present application improves the quality and efficiency of paraffin section slicing by optimizing the slicing process.

[0019] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The above and other objects, features and advantages of the present application will become more apparent through a more detailed description of exemplary embodiments of the present application in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments of the present application.

[0021] Figure 1 This is a schematic diagram / flow chart of Example 1 of the present application;

[0022] Figure 2 is a slice diagram of 2 folds of the heart of M1 (Comparative Example 1);

[0023] Figure 3 is a wrinkle-free slice of M2 heart 1 (Example 1);

[0024] Figure 4 is a cross-section of the fold 2 of the testis of M3 (Comparative Example 2);

[0025] Figure 5 This is a non-wrinkled section of F2 jejunum 1 (Example 1). DETAILED DESCRIPTION

[0026] The embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although the embodiments of the present application are shown in the accompanying drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to make the present application more thorough and complete, and to fully convey the scope of the present application to those skilled in the art.

[0027] Pathological HE staining should be result-oriented rather than process-oriented. In reversing each link of the film making process, it was found that the spreading and scooping of the film is crucial. If there is an error or improper operation in this link, it will directly lead to poor film making or even re-making. Every animal specimen is precious.

[0028] The prior art has the following defects:

[0029] 1. The personnel who spread and retrieve the slides lack the relevant learning background of professional pathological examination technology or medical examination technology. For the spread and retrieve of each tissue slide, they only know how to "use a glass slide to scoop the wax slide in the water onto the glass slide in their hand". They seldom think about how to "fix the point" or "direction", how to retrieve the slides from large tissues, how to retrieve the slides from small tissues, where the tissue patches should be attached, how to deal with slight wrinkles / folds in the tissues, how to standardize the retrieval of slides, etc., all of which can lead to wrinkles.

[0030] 2. The physical fitness, work attitude, environment and psychological factors of the film-picking technicians may also lead to the above reasons, such as distraction, nervousness and lack of confidence when picking up films.

[0031] 3. The slice scooping technician needs to have a good judgment on high-quality slices (the slice scooping technician needs to choose a high-quality slice to scoop up, rather than just picking up the slice that the slicer put down).

[0032] 4. Whether the slicing technician cuts out continuous, complete, flat and wrinkle-free wax slices during the slicing process, and whether the best wax slices are selected and put into the water. The physical fitness, psychological state and environmental factors of the slicer can also lead to poor slicing quality to some extent.

[0033] Therefore, the present application proposes a method for improving the quality of the spreading and scooping techniques of various tissues and organs of rats, and the specific operation is as follows.

[0034] Experimental samples

[0035] Information about the ten animals:

[0036] The animal numbers for female rats were: F1–F5;

[0037] Animal numbers for male rats were: M1–M5;

[0038] Names of rat organs: heart, liver, spleen, lung, kidney, pancreas, adrenal gland, trachea / esophagus, aorta, testicle, seminal vesicle, vagina, ovary, duodenum, rectum, brain.

[0039] Experimental materials: laboratory pure water, pure water measuring instrument, 500mL glass beaker, clean paper, pure black coaster, small curved tweezers, brush, pencil, blade, 10 animal wax blocks to be cut, anhydrous ethanol, 5mL pipette, slide and cover glass, ice tray, neutral gum.

[0040] Experimental instruments: pathology sampling table, fully automatic dehydrator, embedding machine, low-temperature refrigerator, slicer, bleaching machine, slice drying machine, staining machine and sealing machine, double-operated microscope, etc.

[0041] Example 1

[0042] 1) Add a cylinder of water for spreading the slices (cold water bath at room temperature).

[0043] 2) Prepare a 500 mL glass beaker, fill the glass beaker with pure water, use a disposable pipette to absorb 2-3 drops of anhydrous ethanol into the beaker, mix well and place it on the prepared black coaster, and put the above 10 animal wax blocks into a 18-24℃ low-temperature refrigerator for cutting.

[0044] 3) Turn on the slicer power supply, turn on the slicer switch from the lower back corner of the slicer and wait for the self-test to pass and enter the working state;

[0045] Turn on the power of the slide floater, turn on the panel switch, set the water bath temperature to 40-41°C, and set it aside after reaching the temperature;

[0046] Connect the power supply of the drying machine, turn on the panel switch, set the drying temperature to 60-61℃, and set it aside after reaching the temperature;

[0047] Connect the dyeing machine to the power supply, open the water inlet valve, lift the dust cover, remove the covers of each dyeing tank, turn on the dyeing machine, and wait for the self-test to pass before use;

[0048] Turn on the coverslip machine, return the slide loading slot to the position as prompted by the coverslip machine self-test, and start pressurization as prompted after passing. Try it once, and after the glue drips out smoothly, put the glue nozzle in the working position, and the green light will be on. Arrange the clean coverslips and put them into the coverslip slot. Check and add enough neutral gum solution. If there are bubbles in the pipeline, add glue separately until the bubbles are blown out and set aside.

[0049] 4) Take out a piece of animal wax from the refrigerator and place it on the prepared ice tray for cutting.

[0050] 5) Pull the wax block loading rod forward with one hand, hold the wax block with the index finger and thumb of the other hand, put it in, then release the loading rod to clamp the loaded wax block.

[0051] 6) Use the coarse or fine adjustment button to advance or retract the wax block to a position where the blade can cut.

[0052] 7) Set the initial slice thickness to 20-30um, adjust the position to cut out the ideal slice, and rotate the slicer handle clockwise to trim the wax block.

[0053] 8) After trimming until the wax block surface is flat and the tissue surface can be completely cut off, set the thickness of the fine trimmed slice to 2-10um.

[0054] 9) Turn the handle clockwise to slice. Cut a few slices and discard them. When the cut surface is good, start slicing (pay attention to even force when slicing). When slicing, hold the brush in your left hand to guide the slice to stretch it.

[0055] 10) Cut slices continuously and select the two best slices, place them in the glass jar and the slide floater respectively, and start slide floatation.

[0056] 11) Keep the slices in constant temperature water at 40-41℃ (slice bleaching machine) for 2-3 seconds, then remove them and bake them immediately. The baking temperature is 60-61℃ and the baking time is 10-15 minutes.

[0057] 12) The same tissue of the same animal number, for example, the tissue "heart" of the animal number "M1", is cut into two pieces, one of which is placed in a glass beaker and marked as heart ①, and the other is placed in the constant temperature water of the slide floater and marked as heart ②. For example: the heart ① is subjected to the steps of Example 1, that is, there are two steps, firstly, it is placed in a glass beaker and waited for the slide to be fully spread, that is, wrinkles are not visible to the naked eye or wrinkles are flattened in water with small curved tweezers, then it is fished out with a glass slide with the information of the wax slide written on it, and then it is placed in the water of the slide floater for 2-3 seconds, and then it is fished out and the water droplets are gently shaken off by the wrist, and then the slide is immediately baked. The heart ② is subjected to the steps of Comparative Example 1, that is, it is fished out from the constant temperature water of the slide floater, and the water droplets are gently shaken off by the wrist, and then the slide is immediately baked.

[0058] 15) Stain, seal and air dry.

[0059] 16) Microscopic examination, observation of tissue folds and collection of images.

[0060] Principles of microscopic image collection:

[0061] a. Observe and collect the wrinkles and folds of 10 animals under the microscope;

[0062] b. Observe the tissue on the entire slide as a whole, and try to compare and collect any wrinkles, folds, and detachments at the same location of the same tissue;

[0063] c. Classify and name the collected images. For example, M1 heart 2 folded or M1 heart 1 not folded; M1 is the animal number, 1 and 2 represent the above-mentioned Example 1 and Comparative Example 1 respectively)

[0064] d. According to the “Basic Standards for the Quality of Conventional Paraffin-Embedded HE-Stained Sections”, the wrinkles and folds of tissues were judged, and pictures of wrinkles and folds of each tissue were collected for statistical analysis without affecting the diagnosis.

[0065] Notes for the entire example:

[0066] 1. Be proficient in slicing and scooping techniques.

[0067] 2. The water surface of the glass tank and the floater should be kept clean at all times, free of debris and other contamination.

[0068] 3. Keep the brush and small curved tweezers clean.

[0069] 4. The reagents in the staining jars must be within the validity period, and each staining jar must be kept clean and pollution-free. The dye solution must be replaced in time to ensure that the amount of each dye solution is sufficient.

[0070] 5. The slides to be salvaged must be clean and free of contamination, and those that do not meet the requirements must be discarded.

[0071] 6. The tissue wrinkles of all ten animals were observed under a microscope and the specific conditions were recorded immediately.

[0072] Comparative Example 1

[0073] The operation of step 11) in Example 1 is: rinsing in constant temperature water at 40-41°C (slice bleaching instrument) for 2-3 seconds, then taking out and baking the slices immediately. The baking temperature is 60-61°C and the baking time is 10-15 minutes.

[0074] The rest is the same as Example 1.

[0075] Comparative Example 2

[0076] 1. Turn on the power, turn on the power of the film spreading machine from the back, turn on the panel switch, set the water bath temperature to 45℃, and set it aside after reaching the temperature.

[0077] 2. Use a brush in your left hand to gently pick up the slice from the bottom of the cut paraffin slice, transfer it to the water bath for spreading the slices, and let the slice float on the water surface to allow the tissue to unfold naturally.

[0078] 3. When the tissue is fully unfolded, pinch the elbow tweezers, gently touch the separation between the two slices on the water surface with the elbow part, and then quickly release it to break the two connected slices at both ends.

[0079] 4. Hold a labeled slide with the index finger and thumb of your left hand facing up, and insert it into the water next to one end of the slice at an angle of about 30°-45°. Slowly move the slide (or use tweezers to guide the paraffin slice) so that one end of the paraffin slice sticks to the slide, gently lift the slide so that the entire slice is completely attached to the slide, and then remove it from the water. If the sticking is not ideal, float it in the water and stick it again.

[0080] 5. Use clean paper to promptly remove the excess discarded slices in the water bath and put them in a garbage bag.

[0081] 6. Place the pasted glass slides with their front side facing up on the baking machine platform and bake them dry (temperature range 55-70℃, about 15 minutes), then take them out for use. If there are too many glass slides, place the pasted glass slides on the glass slide rack, make the surface level, and place them in a drying oven (temperature range 55-70℃, about 15-30 minutes) to dry them for use.

[0082] 7. In the experiment, it was found that since the water temperature of the floater is constant, the water tension can be adjusted by adjusting the water temperature.

[0083] 8. According to the statistical results of microscopic film reading, both Example 1 and Comparative Example 1 have wrinkles, folds, and film peeling, but Example 1 has fewer wrinkles, folds, and film peeling than Comparative Example 1. The film making effect of Example 1 is better than that of Comparative Example 1 as a whole. Therefore, we recommend using Example 1 as a new method for spreading and lifting films to improve the quality of pathological film making.

[0084] Table 1 shows the microscopic statistics: 10 animals had a total of about 769 HE slides, of which the trachea and esophagus were combined into one slide. In Example 1, the number of wrinkles was 21, the number of folds was 25, and the number of slides that fell off was 0; in Comparative Example 1, the number of wrinkles was 52, the number of folds was 41, and the number of slides that fell off was 2.

[0085] Table 1 Statistical table of the observation of wrinkle degree under microscope by two methods for 110 animals

[0086]

[0087]

[0088] The embodiments of the present application have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A method for preparing slices of rat tissues and organs, characterized in that: The method comprises the following steps: 1) preparing water for spreading the slices, and placing the slices in a cold water bath; the temperature of the cold water bath is room temperature; 2) Place the animal tissue wax block in pure water containing anhydrous ethanol, and then store it at 18-24°C for later use; 3) Place the pre-cooled wax block of animal tissue in 2) on a slicer for Slicing; the initial trimming thickness of the slicer is set to 20-30um; the fine trimming thickness of the slicer is set to 2-10um; 4) screening the slices in 3), and placing the screened slices into a glass jar and a slice bleaching instrument for bleaching; 5) baking the slices after blanching in 4) on a baking machine; the baking time is 10-15 minutes; the baking temperature is 60-61°C.

2. The method according to claim 1, characterized in that: The amount of anhydrous ethanol in the pure water in 2) is as follows: 2-3 drops of anhydrous ethanol are added to 500 mL of pure water.

3. The method according to claim 1, characterized in that: The water bath temperature of the slide floater is 40-41°C.

4. The method according to claim 1, characterized in that: The specific operation steps of slicing in the above 3) are: pull the wax block loading rod forward with one hand, hold the wax block with one hand and then release the loading rod to clamp the loaded animal tissue wax block; adjust the position of the animal tissue wax block to a position that the blade can cut; when slicing, hold the brush with the left hand to guide the slice to stretch it.

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