CN116465708A - Preparation method of kidney tissue specimen - Google Patents

Preparation method of kidney tissue specimen Download PDF

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Publication number
CN116465708A
CN116465708A CN202310561363.1A CN202310561363A CN116465708A CN 116465708 A CN116465708 A CN 116465708A CN 202310561363 A CN202310561363 A CN 202310561363A CN 116465708 A CN116465708 A CN 116465708A
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China
Prior art keywords
specimen
sample
kidney tissue
preparing
embedding
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Inventor
冯强
张惠妮
植海滨
罗海斌
卢真艳
林丽欣
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Guangzhou Huayin Medical Laboratory Center Co Ltd
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Guangzhou Huayin Medical Laboratory Center Co Ltd
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Priority to CN202310561363.1A priority Critical patent/CN116465708A/en
Publication of CN116465708A publication Critical patent/CN116465708A/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
    • G01N1/06—Devices for withdrawing samples in the solid state, e.g. by cutting providing a thin slice, e.g. microtome
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/36—Embedding or analogous mounting of samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/84—Systems specially adapted for particular applications
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
    • G01N23/02—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by transmitting the radiation through the material
    • G01N23/04—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by transmitting the radiation through the material and forming images of the material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
    • G01N23/20—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by using diffraction of the radiation by the materials, e.g. for investigating crystal structure; by using scattering of the radiation by the materials, e.g. for investigating non-crystalline materials; by using reflection of the radiation by the materials
    • G01N23/20008—Constructional details of analysers, e.g. characterised by X-ray source, detector or optical system; Accessories therefor; Preparing specimens therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G01N2001/2873—Cutting or cleaving
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention provides a preparation method of a kidney tissue specimen, which comprises the steps of preparing a light-mirror specimen, an electron microscope specimen and an immunofluorescence specimen of kidney tissue, wherein the preparation method of the light-mirror specimen comprises the following steps: and (3) fixing, dehydrating, paraffin embedding and triple slicing the kidney tissue sample in sequence to obtain the optical microscope sample, wherein the triple slicing refers to three times of slicing the embedded sample along the cross section. The invention creatively adopts triple slices, and the obtained optical lens sample has good preservation, clear structure and good dyeing effect. Compared with the traditional single slice, the method disclosed by the invention has the advantages that the observation rate of the glomerulus is improved to more than 17%, the problem of missing the glomerulus in the traditional method is solved, and meanwhile, the problem of unstable observation effect of the traditional method only in the single slice is solved.

Description

Preparation method of kidney tissue specimen
Technical Field
The invention belongs to the field of biomedicine, and relates to a preparation method of kidney tissue samples, in particular to a preparation method of optical microscope samples (used for optical microscope detection), electron microscope samples (used for transmission electron microscope detection) and immunofluorescence samples (used for fluorescence microscope detection) of kidney tissues.
Background
There are many kinds of kidney diseases, and there are various examination methods, such as urine examination, renal function examination, B-ultrasonic examination, CT examination, etc. The method is characterized in that one or more small kidney tissues are taken from the kidney by a special tool, and classification with different periods and different severity degrees can be obtained through the kidney pathological examination, and particularly, the relation between early clinical symptoms and pathological changes of the kidney tissues in a living body is of great significance for controlling and delaying the progress of various kidney diseases and guiding clinical treatment.
For kidney biopsy specimens, one suitable diagnosis requires a combination of light microscopy, immunofluorescence and electron microscopy. The resulting tissue is then punctured and immediately removed and then visually inspected with an dissecting microscope or with the naked eye to distinguish between cortical and medullary tissue and to determine the presence or absence of glomeruli in the specimen, e.g., the absence of glomeruli, for example, a repackaging may be considered. Appropriate partitioning was performed by observation, contrast, immunofluorescence and electron microscopy.
The kidney biopsy flaking technology plays a vital role in diagnosis of kidney diseases, and clinical experience and related reports of kidney biopsy specimens are combined, so that errors of any step in the flaking technology can influence the accuracy of diagnosis, and therefore, standard technical operation is the basis for ensuring the preparation of good kidney biopsy pathological specimens. At present, the prior art has more mature preparation methods of kidney biopsy tissue specimens, but the methods still have the problems of long specimen preparation time (especially electron microscope specimens), unstable observation effect of the prepared specimens (such as inaccurate observation results on the number of glomeruli in the optical microscope specimens, unstable observation results) and the like.
Therefore, how to simplify the preparation process of the kidney tissue specimen, shorten the preparation time, and ensure and even improve the preparation quality (observation stability and accuracy) of the specimen at the same time becomes a problem to be solved in the field.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a preparation method of a kidney tissue specimen, in particular to a preparation method of a light microscope specimen (used for optical microscope detection), an electron microscope specimen (used for transmission electron microscope detection) and an immunofluorescence specimen (used for fluorescence microscope detection) of kidney tissue.
The preparation method is suitable for kidney biopsy samples and kidney tissues of animals.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for preparing a kidney tissue specimen, the method comprising preparing a light-microscopic specimen, an electron-microscopic specimen and an immunofluorescence specimen of kidney tissue, the method comprising: and (3) fixing, dehydrating, paraffin embedding and triple slicing the kidney tissue sample in sequence to obtain the optical microscope sample, wherein the triple slicing refers to three times of slicing the embedded sample along the cross section.
The prepared optical microscope specimen can be subjected to optical microscope detection after dewaxing and dyeing.
FIG. 1 is a schematic diagram showing the difference between a triple slice of the present invention and a conventional single slice, wherein the conventional single slice technique is to repair the top of a sample to the maximum slice for continuous single-layer slice treatment, and to conduct paper sheets, only the number of glomeruli of the maximum slice can be displayed; the triple slice is formed by trimming the top of the sample, performing one-layer continuous slice, trimming the sample, performing a second-layer continuous slice, trimming the sample, and performing a third-layer continuous slice, wherein all glomeruli can be displayed by the three-layer continuous slice to the greatest extent.
When the optical microscope is used for detecting the contrast sample, the number of glomeruli in the three-layer slice is counted, so that the punctured glomeruli can be observed as much as possible, and missed diagnosis caused by the traditional single-layer slice is avoided (generally, the aperture of a puncture needle is about 700 microns, namely, the diameter of a taken renal tissue sample is about 700 microns, and one glomerulus is about 250 microns, therefore, the triple slice is the most reasonable, and the glomeruli can still be missed by the double slices).
Preferably, the fixing means that the kidney tissue sample is mixed with a fixing solution, and the fixing solution comprises formaldehyde aqueous solution.
Preferably, the procedure of dehydration comprises: treating with 78-82% ethanol solution for 6-10min, 84-86% ethanol solution for 6-10min, 88-92% ethanol solution for 6-10min, 94-96% ethanol solution for 6-10min, anhydrous ethanol for 6-10min, 2 times, toluene for 6-10min, xylene for 8-12min, paraffin for 5-10min, 2 times.
The concentration of the ethanol solution referred to in the present invention is referred to as the volume concentration.
Specific values among the above 78% -82% are, for example, 78%, 79%, 80%, 81%, 82%, etc.
Specific values of 84% -86% are, for example, 84%, 85%, 86% and the like.
Specific values of 88% -92% are as described above, for example 88%, 89%, 90%, 91%, 92% and the like.
The specific value of the above 6-10min is 6min, 6.5min, 7min, 7.5min, 8min, 8.5min, 9min, 9.5min, 10min, etc.
The specific value of the above 8-12min is 8min, 8.5min, 9min, 9.5min, 10min, 10.5min, 11min, 11.5min, 12min, etc.
The specific value of the above 5-10min is, for example, 5min, 5.5min, 6min, 6.5min, 7min, 7.5min, 8min, 8.5min, 9min, 9.5min, 10min, etc.
Preferably, the preparation method of the electron microscope specimen comprises the following steps: and (3) fixing, dehydrating, soaking, embedding and slicing the kidney tissue sample in sequence to obtain the electron microscope specimen.
Preferably, in the preparation of the electron microscope specimen, the fixation refers to the fixation of a kidney tissue sample by mixing with a glutaraldehyde-containing fixing solution.
Preferably, the mass concentration of glutaraldehyde in the fixing solution is 2-4%, e.g., 2%, 2.5%, 3%, 3.5%, 4%, etc.
Paraformaldehyde with a mass concentration of 1% -3% (for example, 1%, 1.5%, 2%, 2.5% and 3%) can also be added into the fixing solution.
Preferably, in preparing an electron microscope specimen, the procedure of dehydration includes: treating with 45% -55% ethanol solution for 2-5min, treating with 65% -75% ethanol solution for 2-5min, treating with 85% -95% ethanol solution for 2-5min, treating with anhydrous ethanol for 2-5min for 2 times, treating with acetone for 2-5min for 2 times.
Specific values in the above 45% -55% are, for example, 45%, 46%, 47%, 48%, 50%, 51%, 52%, 53%, 54%, 55%, etc.
Specific values in the above 65% -75% are, for example, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, etc.
Specific values in the above 85% -95% are, for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, etc.
The specific value of the above 2-5min is 2min, 2.5min, 3min, 3.5min, 4min, 4.5min, 5min, etc.
Preferably, the impregnating comprises: mixing the dehydrated sample with a penetrating agent for 20-50min, wherein the penetrating agent comprises acetone and embedding agent.
Preferably, the mass ratio of the acetone to the embedding agent is (0.5-1.5): 0.5-1.5.
Preferably, the embedding agent comprises an embedding resin (TED pea 18005).
The specific value of the above 20-50min is 20min, 22min, 25min, 27min, 30min, 32min, 35min, 37min, 40min, 45min, 50min, etc.
Specific values in the above range from 0.5 to 1.5 are, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, etc.
Preferably, in preparing the electron microscope specimen, the embedding includes: mixing the soaked sample with embedding agent, and heating to polymerize at 55-65deg.C for 20-40min, and at 80-90deg.C for 1-2 hr.
Specific values of the above 55-65deg.C include 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, etc.
The specific value of the above 20-40min is 20min, 22min, 25min, 27min, 30min, 32min, 35min, 37min, 40min, etc.
Specific values of the above 80-90deg.C include 80 ℃, 81 ℃, 82 ℃, 83 ℃, 84 ℃, 85 ℃, 86 ℃, 87 ℃, 88 ℃, 89 ℃, 90 ℃, etc.
The specific value in the above 1-2h is 60min, 65min, 70min, 75min, 80min, 85min, 90min, 95min, 100min, 105min, 110min, 115min, 120min, etc.
The method specifically designs parameters of a dehydration procedure, soaking and embedding (heating polymerization) treatment aiming at the kidney tissue sample, and the parameters are mutually matched, so that the effect of not reducing or even improving the flaking quality is realized under the condition of greatly improving the preparation speed of the sample (shortening about 34h to about 4h in the traditional method from dehydration to embedding step). The quality of the obtained sheet can reach the standard by adopting the parameters in the range of the invention to operate, and the quality of the sheet obtained by adopting the preferred scheme in the embodiment of the invention is optimal.
Preferably, the preparation method of the immunofluorescence specimen comprises the following steps: and mixing the kidney tissue sample with the OTC embedding agent, cutting the ice PAS slice into two surfaces, observing whether glomeruli exist under a light microscope, continuing to deeply observe when glomeruli do not exist and the tissue is cortex, ensuring that the glomeruli are surely cut, adding an antibody into the slice with the glomeruli, and sealing the slice with the glomeruli to obtain the immunofluorescence specimen.
The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.
For a kidney biopsy tissue specimen, a proper diagnosis needs to combine a light mirror, immunofluorescence and an electron microscope, and compared with the prior art, the invention has the following beneficial effects:
(1) In the preparation of the optical lens specimen, the invention creatively adopts the triple slice, and the obtained optical lens specimen has good preservation, clear structure and good dyeing effect. Compared with the traditional single slice, the method disclosed by the invention has the advantages that the observation rate of the glomerulus is improved to more than 17%, the problem of missing the glomerulus in the traditional method is solved, and meanwhile, the problem of unstable observation effect of the traditional method only in the single slice is solved.
(2) In the preparation of the electron microscope specimen, the invention realizes the effect of not reducing or even improving the sheeting quality under the condition of greatly improving the specimen preparation speed (shortening about 34h to about 4h in the traditional method from dehydration to embedding step) through the design of the whole flow and the cooperation of all parameters.
(3) In the preparation of immunofluorescence specimens, the fluorescence sections of the invention are different from the traditional 'blind cutting', each example can be seen under a lens, and ice PAS is added to ensure that glomerulus is cut necessarily.
Drawings
FIG. 1 is a schematic diagram showing a comparison of a triple slice of the present invention with a conventional single slice.
FIG. 2 is a view showing the observation result of an electron microscope specimen prepared in the example, wherein the lower right corner scale is 10. Mu.m.
FIG. 3 is a diagram showing the observation results of the electron microscope specimen prepared in the comparative example, with a lower left corner scale of 10. Mu.m.
FIG. 4 is a graph showing the HE staining results of the optical samples prepared in the examples.
FIG. 5 is a graph showing the HE staining results of the optical samples prepared in the comparative examples.
FIG. 6 is a graph showing the PASM staining results of the optical lens samples prepared in the examples.
FIG. 7 is a graph showing the PASM staining results of the optical lens specimens prepared in the comparative example.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
In the following examples, all reagents and consumables were purchased from the reagent manufacturers routine in the art unless specifically indicated; unless otherwise indicated, all methods and techniques used are those conventional in the art.
Examples
The embodiment provides a preparation method of a kidney tissue specimen, which specifically comprises the following steps:
firstly, after the kidney tissue sample is obtained, the sample is immediately segmented and is placed into a bottle corresponding to a light mirror, immunofluorescence and electron microscope which are filled with different preservation solutions for fixation. After the sample arrives in the laboratory, the light mirror, the immunofluorescence and the electron microscope sample are divided into three groups of experimenters, and three lines are synchronously manufactured, so that waiting time is saved, and the three preparation routes are as follows:
(1) Preparation of optical lens specimen
1. Fixing and drawing materials: the obtained kidney tissue sample is fixed in a fixing solution (10% neutral formaldehyde solution, xielong science 83012) at room temperature for 45min to be dehydrated, after the fixing, whether the sample bottles are arranged in sequence from small size to large size is checked, after the sample is clamped in embedding paper, the sample is folded diagonally until the sample cannot fall out, the cover is tightly covered in an embedding box with corresponding pathological number, the length of the registered tissue is soaked in 80% ethanol, and the embedding box is checked for a plurality of times when the materials are obtained, whether the pathological number of the tissue is consistent with that of the tissue and whether the tissue in the sample bottle is taken out.
2. Dehydrating: 80% ethanol for 8 min-85% ethanol for 8 min-90% ethanol for 8 min-95% ethanol for 8 min-absolute ethanol for 10 min-xylene for 8 min-xylene for 10 min-paraffin for 5 min-paraffin for 10 min.
3. Embedding: placing the dehydrated embedding box into paraffin of the embedding machine after dehydration, using a paraffin mold with a corresponding size, injecting molten paraffin (Vigers-high-quality pathological paraffin-2200046) into the paraffin mold, clamping dehydrated tissues into the paraffin mold, checking whether the length of the dehydrated tissues is consistent with the length of the dehydrated tissues registered in the registered book, placing the dehydrated tissues on a small freezing table for solidification after selecting an embedding surface, slightly pressing tweezers to enable the tissues to be in the paraffin and cling to the bottom of the paraffin mold, and injecting a proper amount of molten paraffin to be placed on the freezing table for solidification after placing the embedding box. Demolding after the wax is completely solidified, and trimming the periphery of the wax block.
4. Paraffin section (triple section): and clamping the solidified and demoulded wax blocks into a groove of a slicing machine, adjusting the thickness of the slicing machine to 5 mu m, trimming the wax blocks, taking down the wax blocks on a freezing table and the like after cutting out the largest surface of the tissue, adjusting the thickness of the slicing machine to 1.5 mu m after trimming the wax blocks, and performing triple slicing.
The cut wax sheet is gently clamped by forceps and placed on the normal-temperature water surface for separation, the wax sheet with uncomfortable thickness is removed, the wax sheet is fished out to be on the water surface at 50 ℃ to flatten tissues, and then the wax sheet is fished out to be at the center of a glass slide. If the paraffin fluorescence is needed, the slice is taken out and then whether glomerulus exists is observed under a microscope. And then putting the fished slide into a corresponding slide frame for baking until paraffin is dissolved.
5. Dewaxing dyeing sealing piece:
dewaxing: putting the baked slices into xylene for dewaxing, putting into absolute ethyl alcohol three cylinders after each cylinder is 5 minutes after three cylinders of xylene are used for each cylinder for 1 minute, putting into 95% ethyl alcohol and taking out for one minute after one minute for flushing;
dyeing: HE staining: (1) washing with water, dyeing with hematoxylin for 4-5 min (removing oxide film on the surface of hematoxylin dye liquor before dyeing), washing with water, differentiating with 1% hydrochloric acid alcohol for 3-5s, and returning running water to blue (warm water to blue when the temperature is low in winter), and observing under a mirror; -eosin 7-8 min-85% ethanol-95% ethanol-absolute ethanol-xylene-tablet;
(2) note that: eosin is an alcohol-soluble dye, and the alcohol soaking time is not too long in the dehydration process.
(2) Preparation of electron microscope specimen
1. The obtained kidney tissue sample is placed in 2.5% glutaraldehyde preservation solution for fixation for 1h (4 ℃), then clamped out and placed on absorbent paper, and glutaraldehyde solution on the surface of the sample is sucked dry. Sequentially placing the sample baskets into a KOS multifunctional microwave tissue processor, and setting the parameters of the microwave tissue processor as shown in Table 1.
TABLE 1
1 Pb 6’
2 Pb 6’
3 BUFFER RINSE 2 6’
4 Pb 8’
5 MW OSMIUM 11’35”
6 Water Rinse 1 4’20”
7 Water Rinse 2 4’40”
2. Wash four times with 0.1M PB for 5 minutes each.
3. The sample was fixed with 1% aqueous osmium acid for 15 minutes.
4. Wash three times with 0.1M PB for 5 minutes each.
5. Dehydration procedure:
5.1, treating with 50% ethanol for 3 min;
5.2, treating with 70% ethanol for 3 min;
5.3, treating with 90% ethanol for 3 min;
5.4, treating the mixture with absolute ethyl alcohol for 3 minutes;
5.5, treating the mixture with absolute ethyl alcohol for 3 minutes;
5.6, 100% acetone treatment for 3 min;
5.7, 100% acetone for 3 min.
6. Impregnation and embedding procedure
6.1, acetone: pure resin embedding agent (embedding resin, TED pea 18005) =1:1 (weight ratio) soak the sample for 30 minutes.
6.2 pure resin embedding
A. Placing the embedding plate with the corresponding pathological number in a baking oven at 37 ℃ for 15 minutes;
B. placing pure resin embedding agent (embedding resin, TED PELLA 18005) into 37 deg.C oven, baking and thawing, and keeping resin dry for 15min
C. Uniformly filling pure resin embedding agent on the embedding plate;
D. embedding the sample in the embedding plate at the position corresponding to the pathological number;
E. heating and polymerizing:
a 60 ℃ incubator for 0.5 hours;
the temperature of 85 ℃ is 1.5 hours.
7. Ultrathin section
7.1, set control board
7.2, rough repair of resin Block sample
A. Placing the block repairing seat in the tool apron area, then placing the sample head on the block repairing seat, and locking the sample head and the block repairing seat;
B. fixing the sample on a sample head, and locking the sample by using a hexagonal screw;
C. the top end of the embedding block is flattened according to the position of the sample, and tissues are exposed;
D. cutting off surrounding embedding agent and related parts, trimming the part to be observed into a cone, taking care that the cone is not trimmed to be protruded, otherwise, easily generating vibration marks in the slicing process;
E. the plane of the top end is trimmed to be a flat trapezoid or square with obvious boundary, and a corner is trimmed for positioning if necessary.
7.3 semi-thin slice and positioning
A. The sample head and the block repairing seat are taken down together;
B. placing the sample head on the sample rod and locking;
C. an illumination lamp connecting the sample head and the tool apron region;
D. placing the tool apron in the tool apron area and locking;
E. placing the sample on a sample head and locking the sample by using a hexagonal screw;
F. placing the glass cutter on a cutter seat, locking the glass cutter, adjusting the slicing angle of the glass cutter through a clearance angle, and fixing the glass cutter on the proper position, wherein the cutting angle is generally 6 scales;
G. firstly, adjusting the position of a microscope and the position of a knife to enable the microscope to see the cutting edge and the position of a sample block, adjusting the microscope to the maximum magnification, and adjusting the position of a sample surface to enable two parallel edges of the cutting surface to be parallel to the cutting edge;
H. rotating the manual knobs on the tool apron, selecting a tool edge meeting the requirements, turning off top light and spot light, and only turning on back light;
I. coarse-tuning feeding, namely, performing coarse-tuning feeding by using a handwire on the left hand until a shadow (reflection image) is observed on the surface of a tissue block, carefully observing the shadow, adjusting the angle between a cutting edge and a sample surface, enabling the cutting edge to be approximately parallel to the sample surface, manually performing coarse-tuning, leveling the sample surface, rotating a manual knobs on a cutter holder, and selecting a new cutter edge;
J. the thickness of the semi-thin slice is set to be 1500nm, and an automatic slicing switch (M key of a controller) can be turned on to slice the slice;
K. the sections were transferred to a slide for staining for viewing under a light microscope;
l, further repairing the embedded block according to the observation under the light mirror and the azimuth mark on the semi-sheet;
and M, reserving a part to be observed by an electron microscope according to the block repairing method, and continuing to repair the rest part to remove redundant embedded blocks until the part is completely repaired.
7.4 ultrathin section
A. The glass cutter is taken off from the cutter holder, and the diamond cutter is placed on the cutter holder and locked;
B. firstly, adjusting the position of a microscope and the position of a knife to enable the microscope to see the cutting edge and the position of a sample block, adjusting the microscope to the maximum magnification, and adjusting the position of a sample surface to enable two parallel edges of the cutting surface to be parallel to the cutting edge;
C. rotating the manual knobs on the tool apron, selecting a tool edge meeting the requirements, turning off top light and spot light, and only turning on back light;
D. coarse-tuning feeding, namely, performing coarse-tuning feeding by using a handwire on the left hand until a shadow (reflection image) is observed on the surface of a tissue block, carefully observing the shadow, adjusting the angle between a blade and a sample surface to enable the blade to be completely parallel to the sample surface (the shadow is parallel at the moment and the thickness of the shadow passing through the range of the sample surface is unchanged when the sample block moves up and down), and finally performing fine-tuning feeding to enable the shadow to be a slit which is almost imperceptible;
E. after the tool is set, the top light and the spot light are turned on, so that double distilled water can be injected into the tool groove until a bright surface of reflected light appears on the liquid surface;
F. after water is added, an automatic slicing switch (M key of a controller) can be turned on to slice, and generally, silvery white slices (60-80 nm) are required to be cut;
G. fishing: the copper net is clamped by forceps, the slice aligned to the liquid surface is lightly dipped (when the supporting film is arranged, the film surface is downward), the slice is covered on the copper net, and then the slice is dried.
In addition, the copper net can be clamped by forceps and immersed in the water tank, the position of the slicing bar is fixed by eyelashes or a steel needle or one corner of the slice is lightly attached to one end of the copper net, and then the copper net is lifted from bottom to top.
(3) Preparation of immunofluorescence specimen
1. Sample cleaning: washing a sample by using fluorescent sample washing liquid, filling the fluorescent sample washing liquid into a sample bottle filled with the sample for the first time, standing for 2 minutes, sucking out the sample washing liquid, adding the fluorescent sample washing liquid for the second time (two thirds), and standing for 4 minutes.
2. Adding a write sheet of the project: and (3) writing a written slide according to the specimen application form and the special hospital requirements, and placing the written slide on a conventional slide with a corresponding number.
3. Sampling a sample: a small drop of frozen section embedding agent OCT (Japanese cherry-OCT frozen embedding agent-4583) and a piece of clean absorbent paper are prepared on the paperboard for writing the corresponding sample bottle number while the sample is kept still, the kept sample is clamped by forceps and placed on the absorbent paper, the surface water is sucked dry, and then the sample length is recorded in the OCT of the corresponding numbered paperboard.
4. Freezing and slicing: when the slice is cut, the number of the cut sample is required to be consistent with that of the slide, whether glomerulus exists or not is observed under a rear mirror of the two surfaces of the ice PAS, when glomerulus does not exist and the tissue is cortex, deep observation is continued until the tissue is cut to the maximum surface, and the slice is cut until the glomerulus does not exist, and then the slice is cut, and the slice is marked with an asterisk to be subjected to paraffin fluorescence. Deep repair tissue is cut into pieces when glomeruli are not present and the glomeruli are medulla, and paraffin fluorescence is carried out when the glomeruli are not present after observation; after the glomerulus was cut and applied as a slide, it was observed whether the glomerulus remained. The front surface of the glass slide corresponding to the tissue uses the circle closed by the fluorescence combined strokes, and the back surface uses a marker pen to carry out circling positioning.
5. Adding an antibody: the frozen fluorescence is obtained by washing the circled slide with three cylinders of PBS, spin-drying, adding corresponding antibody, placing into a light-shielding box, and adding a bit of water into the light-shielding box to prevent the oven from evaporating the antibody on the slide. Placing the glass slide in a 39 ℃ oven for timing, adding the first antibody for 33 minutes, placing the second antibody into the 39 ℃ oven for 51 minutes, taking back, flushing the antibody on the glass slide with small water flow, flushing with PBS, spin-drying, adding the second antibody into the 39 ℃ oven for 33 minutes. After the paraffin fluorescent dewaxed primary antibody is circled, the paraffin fluorescent dewaxed primary antibody is rinsed by PBS, added with proteinase K and put into a 39-degree oven for antigen retrieval for 31 minutes, and then the corresponding operation is carried out according to the frozen fluorescent primary antibody. The secondary antibody is subjected to high-temperature high-pressure repair, distilled water is added into an autoclave according to the ratio of 8.0EDTA to distilled water of 1:100, the electromagnetic oven is regulated to be at 5 gear for 6 minutes after the autoclave is inflated, the electromagnetic oven is kept stand for 5 minutes after the timing is finished, the secondary antibody is flushed for 5 minutes after the timing is finished, the glass slide subjected to high-temperature high-pressure repair is taken out, cleaned and circled, and then the corresponding operation is carried out according to the freezing secondary antibody method
6. Sealing piece swinging piece: sealing piece: the slide with the reached time is taken back from the oven, washed by small water flow, washed by PBS, and put into a slide plate after the water on the back of the slide is wiped off, and two glycerol seal tablets are dropped on the slide plate to take a cover glass. Swinging piece: slide with corresponding addition item of the pathological number is put on according to the addition registered on the specimen registry from small size to large size. The doctor is informed to read the slide after the laid slide is placed beside the fluorescence microscope in a dark place.
Comparative example-traditional preparation method of kidney biopsy specimen
(1) Preparation of optical lens specimen
The difference from example (1) is only that a single slice was performed in paraffin sections, and other operations are described in example 1.
(2) Preparation of electron microscope specimen
Fixing: immediately placing the sampled sample into 2.5% glutaraldehyde, fixing for 1h in a refrigerator at 4 ℃, adding 0.05mol/L phosphoric acid buffer solution for washing for 3h, and then adding 1% osmium acid for fixing for 1h at room temperature;
dehydrating: sequentially dehydrating with 50% ethanol, 65% ethanol, 75% ethanol, 85% ethanol, 95% ethanol and anhydrous ethanol for 30 min/time;
soaking: 3 times of soaking the oxidized acrylic acid for 20 min/time; solidifying to polymerize and harden the mixture to form a light yellow transparent block;
embedding pure resin:
A. placing the embedding plate with the corresponding pathological number in a baking oven at 37 ℃ for 15 minutes;
B. placing the pure resin embedding agent into a baking oven at 37 ℃ for baking and thawing, and keeping the resin dry for 15 minutes;
C. uniformly filling the embedding plate with an embedding agent;
D. embedding the sample in the embedding plate at the position corresponding to the pathological number;
E. heating and polymerizing:
a 37 ℃ incubator for 3 hours;
a 45 ℃ incubator for 1 hour;
the temperature was kept at 60℃for 24 hours.
The slicing step is described in example (2) and will not be described in detail.
Comparison of sample preparation effects
The same kidney tissue sample is divided, corresponding samples including a light-mirror sample, an electron microscope sample and an immunofluorescence sample are prepared according to the methods of the above examples and comparative examples, and then the samples are detected by the same instrument and under the same environment, and photographed, and the results are shown in fig. 2-7.
And (3) the observation result of the electron microscope shows that: compared with the traditional method, the electron microscope specimen prepared by the method has the advantages of clearer observation structure and better sheeting quality. Namely, the method of the invention realizes the effect of not reducing or even improving the sheeting quality under the condition of greatly improving the specimen preparation speed.
The observation result of the light mirror shows that the light mirror sample prepared by the method has good preservation, clear structure and good dyeing effect.
In addition, samples were prepared by the methods for preparing the optical glass samples of examples and comparative examples, and the observed number of glomeruli was compared, and the results showed that 1560 glomeruli were detected in total by the method of comparative example (single slice) in 100 reports, and 15.6 glomeruli were detected on average; a total of 1828 glomeruli were detected using the method of the example (i.e., triple slice), an average of 18.28; conclusion: compared with single-layer slices, the triple slices have the advantage that the lifting rate of glomeruli is 17.18 percent, which is obvious.
The applicant states that the present invention is described by way of the above examples as a method for preparing a kidney tissue specimen according to the present invention, but the present invention is not limited to the above examples, i.e. it is not meant that the present invention must be practiced in dependence upon the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Claims (10)

1. A method for preparing a kidney tissue specimen, the method comprising preparing a microscope specimen, an electron microscope specimen and an immunofluorescence specimen of kidney tissue, the method comprising: and (3) fixing, dehydrating, paraffin embedding and triple slicing the kidney tissue sample in sequence to obtain the optical microscope sample, wherein the triple slicing refers to three times of slicing the embedded sample along the cross section.
2. The method of claim 1, wherein the fixing comprises mixing the kidney tissue sample with a fixing solution, and the fixing solution comprises an aqueous formaldehyde solution.
3. The method of preparing a renal tissue sample according to claim 1 or 2, wherein the procedure for dehydration comprises: treating with 78-82% ethanol solution for 6-10min, 84-86% ethanol solution for 6-10min, 88-92% ethanol solution for 6-10min, 94-96% ethanol solution for 6-10min, anhydrous ethanol for 6-10min, 2 times, toluene for 6-10min, xylene for 8-12min, paraffin for 5-10min, 2 times.
4. A method of preparing a kidney tissue specimen according to any one of claims 1-3, wherein the method of preparing an electron microscope specimen comprises: and (3) fixing, dehydrating, soaking, embedding and slicing the kidney tissue sample in sequence to obtain the electron microscope specimen.
5. The method according to claim 4, wherein the fixing means mixing the kidney tissue sample with a glutaraldehyde-containing fixing solution for fixing.
6. The method for preparing a kidney tissue specimen according to claim 5, wherein the mass concentration of glutaraldehyde in the fixing solution is 2 to 4%.
7. The method of preparing a kidney tissue specimen according to any one of claims 4 to 6, wherein in preparing an electron microscope specimen, the procedure of dehydration comprises: treating with 45% -55% ethanol solution for 2-5min, treating with 65% -75% ethanol solution for 2-5min, treating with 85% -95% ethanol solution for 2-5min, treating with anhydrous ethanol for 2-5min for 2 times, treating with acetone for 2-5min for 2 times.
8. The method of preparing a kidney tissue specimen according to any of claims 4-7, wherein said impregnating comprises: mixing the dehydrated sample with a penetrating agent for 20-50min, wherein the penetrating agent comprises acetone and an embedding agent;
preferably, the mass ratio of the acetone to the embedding agent is (0.5-1.5): 0.5-1.5;
preferably, the embedding agent comprises a resin.
9. The method of preparing a kidney tissue specimen according to any one of claims 4 to 8, wherein in preparing an electron microscope specimen, the embedding comprises: mixing the soaked sample with embedding agent, and heating to polymerize at 55-65deg.C for 20-40min, and at 80-90deg.C for 1-2 hr.
10. The method of preparing a kidney tissue specimen according to any one of claims 1 to 9, wherein the method of preparing an immunofluorescent specimen comprises: and mixing the kidney tissue sample with the OTC embedding agent, cutting the ice PAS slice into two surfaces, observing whether glomeruli exist under a light microscope, continuing to deeply observe when glomeruli do not exist and the tissue is cortex, ensuring that the glomeruli are surely cut, adding an antibody into the slice with the glomeruli, and sealing the slice with the glomeruli to obtain the immunofluorescence specimen.
CN202310561363.1A 2023-05-18 2023-05-18 Preparation method of kidney tissue specimen Pending CN116465708A (en)

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