CN116718452A - Application method of frozen section in Golgi staining - Google Patents

Application method of frozen section in Golgi staining Download PDF

Info

Publication number
CN116718452A
CN116718452A CN202310780327.4A CN202310780327A CN116718452A CN 116718452 A CN116718452 A CN 116718452A CN 202310780327 A CN202310780327 A CN 202310780327A CN 116718452 A CN116718452 A CN 116718452A
Authority
CN
China
Prior art keywords
solution
brain tissue
distilled water
5min
frozen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202310780327.4A
Other languages
Chinese (zh)
Inventor
夏白娟
向阳
李容瑢
尹丹
李一欣
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guizhou Medical University
Original Assignee
Guizhou Medical University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guizhou Medical University filed Critical Guizhou Medical University
Priority to CN202310780327.4A priority Critical patent/CN116718452A/en
Publication of CN116718452A publication Critical patent/CN116718452A/en
Pending legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • G01N1/42—Low-temperature sample treatment, e.g. cryofixation
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Sampling And Sample Adjustment (AREA)

Abstract

The invention provides a method for applying frozen sections in Golgi staining, which comprises the following steps: drawing materials, immersing in a cryoprotectant, freezing, slicing, dyeing, dehydrating and installing. The invention is simple and practical and is easier to operate.

Description

Application method of frozen section in Golgi staining
Technical Field
The invention belongs to the technical field of Golgi staining, and particularly relates to an application method of a frozen section in Golgi staining.
Background
The existing method for embedding tissue slices and patches by double distilled water in the frozen slices in Golgi staining is complex, so that the repeated difficulty is caused, the requirements are strict, otherwise, the later slice falling phenomenon is serious, the tissue shrinkage is caused due to dehydration and transparency, the slice is not good, and the method after slice falling is not solved at present; some commercial kits have short guidance on frozen sections, but are expensive and not easy to popularize; there are also paraffin sections and vibration section methods, but since the paraffin section thickness is less than 60 μm, dendrite branching and axon analysis cannot be performed well, and vibration microtomes are expensive and have a problem of difficulty in popularization. Thus, there remains a need to create a simpler, more practical, and easier to handle golgi-kox dyeing scheme.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for applying the frozen section to Golgi staining, which is simple, practical and easy to operate, aiming at the defects of the prior art.
In order to solve the technical problems, the invention adopts the following technical scheme: a method of using frozen sections in golgi staining, the method comprising:
s1, material selection: taking brain tissue of a C57BL/6J mouse, washing with double distilled water, cutting into blocks, and cutting into brain tissue blocks with the thickness of 0.5 cm;
s2, immersion liquid: soaking the brain tissue block obtained in the step S1 in a centrifuge tube filled with working solution for 6-24 hours, replacing the working solution, and keeping the soaked brain tissue block at room temperature in a dark place without replacing the working solution after the soaking for 7-14 days;
the preparation method of the working solution comprises the following steps: vibrating and uniformly mixing the solution A, the solution B, the solution C and double distilled water according to the volume ratio of 5:5:4:10, reacting for 24-48 hours in the dark at room temperature, and filtering by a 0.22 mu mm filter or filter paper to obtain working solution;
the solution A is prepared by dissolving 5g of potassium dichromate in 100mL of double distilled water;
the solution B is prepared by heating 5g of mercury chloride in a water bath at 90 ℃ and dissolving in 100mL of double distilled water;
the solution C is prepared by dissolving 5g of potassium sorbate in 100mL of double distilled water;
s3, soaking the cryoprotectant:
taking out the brain tissue blocks after immersion, which are obtained in the step S2, by using a plastic clamp under the illumination environment of 100Lux, wiping the brain tissue blocks with absorbent paper, then putting the wiped brain tissue blocks into a cryoprotectant for soaking for 2-3 days, and replacing the cryoprotectant every 24 hours until the brain tissue blocks are submerged, thus obtaining the brain tissue blocks after soaking the cryoprotectant;
the cryoprotectant is prepared from the following raw materials in parts by weight: 20g of sucrose and 15-20 mL of glycerol, and the volume is fixed to 100mL by double distilled water;
s4, freezing and slicing:
s401, dropwise adding an OCT embedding agent into a precooled tray in a frozen microtome at the temperature of minus 25 ℃ to minus 20 ℃ to form an ice bottom, fixing the brain tissue block soaked with the cryoprotectant liquid obtained in the S3 on the tray, dropwise adding the OCT embedding agent into the brain tissue block soaked with the cryoprotectant liquid, and freezing in the frozen microtome for more than 30 minutes to obtain a frozen brain tissue block;
s402, slicing the brain tissue block frozen in the S401 by using the frozen microtome, clamping the edge by using forceps after slicing, and placing the frozen brain tissue block into a pore plate filled with 30% sucrose solution for expanding to obtain slices;
the 30% sucrose solution is prepared by dissolving 30g sucrose with 100mL double distilled water;
s403, sucking 30% sucrose solution on a glass slide hung with gelatin by using a Pasteur pipette to form a water area, moving the slice obtained in S402 on the glass slide by using a frozen slice blade, flattening the slice, discarding the redundant sucrose solution, and then adjusting the position of the slice by pulling a needle or moving tissues into a dish filled with 30% sucrose solution for pulling out the slice so that the slice is attached to the glass slide hung with gelatin, thereby obtaining attached slices;
s404, placing the stuck pieces obtained in the S403 in a dark place, standing overnight at room temperature under a ventilation condition or baking at 37 ℃ for 30-60 min to obtain dried pieces;
s5, dyeing:
s501, washing the dried slices obtained in the S404 with double-steaming water for 2 times, each time for 5min;
s502, dyeing in a dyeing tank filled with ammonia solution for 10min in a dark place, and washing with double-distilled water for 2 times, wherein each time lasts for 5min;
the ammonia solution is prepared from ammonia water and double distilled water with the mass fraction of 25% -28% in a volume ratio of 3:1;
s503, washing the film in a 1% sodium thiosulfate solution for more than 15 min;
s504, after film washing, placing the film on a frame, horizontally placing the frame in double distilled water, and washing twice for 5min each time with the organized surface upwards;
s505, placing the flakes in an oven with the temperature of 60 ℃ and baking for 2-3 hours in a dark place;
s506, washing for 4 times, each time for 5min;
s6, dehydration and installation:
s601, dehydration and transparency: the stained slide obtained in S506 was placed in the following solutions in order, and dehydrated and transparent: 5min of ethanol aqueous solution with the mass fraction of 50%, 5min of ethanol aqueous solution with the mass fraction of 75%, 5min of ethanol aqueous solution with the mass fraction of 95%, 15min of absolute ethanol A, 25min of absolute ethanol A, 5min of dimethylbenzene I and 5min of dimethylbenzene II;
s602, sealing the film by using neutral resin, and storing the film in the dark until microscopic examination.
Preferably, the well plate in S402 is a 6-well plate or a 12-well plate.
Compared with the prior art, the invention has the following advantages:
the invention adopts the OCT embedding method, thereby reducing the difficulty of embedding by ice. The problems of limited use time of the frozen microtome and the like exist in many laboratories at present, the problem is solved by introducing a slice fishing method, and experiments show that the 12-pore plate or the 6-pore plate can be used for expanding slices well and carrying and moving well; the patch method in the prior art has the advantages of large difficulty, multiple processes and complex operation, is simpler and easier to handle, and provides a method for solving the problem of chip dropping and a method for processing after chip dropping in the follow-up process; the dyeing method shortens the staged washing time, and adds the step of baking after dyeing, so that the problem of subsequent piece falling can be fully solved, but the dyeing effect is not affected.
The invention is described in further detail below with reference to the drawings and examples.
Drawings
Fig. 1 is a sheet development process of embodiment 1 of the present invention.
Fig. 2 is a patch process and drying criteria of example 1 of the present invention.
FIG. 3 is a tissue staining protocol of example 1 of the present invention.
FIG. 4 is the Golgi staining results of example 1 of the present invention.
Detailed Description
Example 1
The method for applying the frozen section in the Golgi staining in the embodiment comprises the following steps:
s1, material selection: taking brain tissue of a C57BL/6J mouse, washing with double distilled water, cutting into blocks, and cutting into brain tissue blocks with the thickness of 0.5 cm;
s2, immersion liquid: soaking the brain tissue block obtained in the step S1 in a centrifuge tube filled with working solution for 24 hours, replacing the working solution, and keeping the soaked brain tissue block at room temperature in a dark place without replacing the working solution for 14 days;
the preparation method of the working solution comprises the following steps: vibrating and uniformly mixing the solution A, the solution B, the solution C and double distilled water according to the volume ratio of 5:5:4:10, reacting for 24 hours in the dark at room temperature, and filtering by a 0.22 mu mm filter to obtain a working solution;
the solution A is prepared by dissolving 5g of potassium dichromate in 100mL of double distilled water;
the solution B is prepared by heating 5g of mercury chloride in a water bath at 90 ℃ and dissolving in 100mL of double distilled water; s3, soaking the cryoprotectant:
taking out the brain tissue block after immersion obtained in the step S2 by using a plastic clamp under the illumination environment of 100Lux, wiping the brain tissue block with absorbent paper, immersing the wiped brain tissue block in a cryoprotectant for 2 days, slightly acting (the brain tissue immersed by the working solution is particularly fragile), replacing the cryoprotectant every 24 hours, and slicing until the brain tissue block is immersed into the cryoprotectant, thereby obtaining the brain tissue block immersed by the cryoprotectant;
the cryoprotectant is prepared from the following raw materials in parts by weight: 20g of sucrose and 15mLL of glycerol, and using double distilled water to fix the volume to 100mL;
s4, freezing and slicing:
s401, dropwise adding an OCT embedding agent into a precooled tray in a frozen microtome at the temperature of minus 20 ℃ to form an ice bottom, fixing the brain tissue block soaked with the cryoprotectant obtained in the S3 on the tray, dropwise adding the OCT embedding agent onto the brain tissue block soaked with the cryoprotectant, forming an adhesion layer of about 2mm around the brain tissue, and freezing for 30min in the frozen microtome to obtain a frozen brain tissue block;
the OCT embedding method reduces the difficulty of embedding with ice, is rare in frozen sections, is likely to cause ice fragmentation after being embedded by a disposable embedding dish, further influences the sections, has poor control on strength during the sections, causes ice fragmentation of partial areas during the sections, exposes partial surrounding tissues, leads to tissue shedding due to excessive exposure, takes a certain time to grasp the section skills, and has higher difficulty in the hands;
s402, slicing the brain tissue block frozen in S401 by using the frozen microtome, as shown in FIG. 1: a, after slicing, the edges are clamped by forceps and placed into a 6-well plate containing 30% sucrose solution for spreading, and the well plate is shown in FIG. 1: b, slightly waving the tissue by using forceps to help the tissue to spread sheets so as to obtain the sections; as in fig. 1: c is shown as the specification; when slicing, if the tissues are too sticky to be spread, the freezing time should be prolonged, if the slices are rigid, the time for soaking the cryoprotectant liquid should be prolonged, and whether the cryoprotectant liquid is qualified or not should be detected; FIG. 1 is a slide-out process wherein A represents tissue obtained after a microtome cut; b, an orifice plate filled with sucrose with a proper volume; c: representing the tissue after the film is spread, wherein the tissue is smooth, flat and complete;
the 30% sucrose solution is prepared by dissolving 30g sucrose with 100mL double distilled water;
s403, sucking 30% sucrose solution on the glass slide hung with gelatin by using a Pasteur pipette to form a water area as shown in FIG. 2: c, moving the slice obtained in S402 onto a glass slide with a frozen slicing blade, flattening the slice, and discarding the excess sucrose solution, as shown in FIG. 2: B. c, D and E, then the needle is moved to adjust the slicing position, and tissues can be moved into a dish filled with 30% sucrose solution for slicing so that the slices are attached to a glass slide hung with gelatin, and then attached slices are obtained; as in fig. 2: a and E are shown;
the embodiment uses the slice dragging method, ensures that the slice is not required to be immediately pasted, is convenient to carry and store, ensures that the service time of the frozen slicer can be used for slicing, does not waste time due to pasting, and solves the problem that the service time of the frozen slicer is limited.
S404, placing the attached pieces obtained in the S403 in a dark place, and standing overnight at room temperature under a ventilation condition to obtain dried pieces; as shown in fig. 2: f, the baking can be stopped until the tissue on the sheet has dried, as shown by the two left arrows in fig. 2:G, and if too long, the tissue will crack, destroying the tissue integrity, as shown by the two right arrows in fig. 2:G;
in the step, the pasted flakes can be placed in a dark place and baked at 37 ℃ for 30-60 min to obtain dried flakes;
FIG. 2 is a diagram of the process of pasting and drying criteria; wherein A is a fishing piece in a dish; b, scooping tissue from the pore plate; c: representing the process of moving tissue with a blade into the water of a gelatin-mounted slide; d: representing flattening of tissue on a slide; e: representing the attached tissue picture; f, representing the dried tissue; g: represents a comparison of tissue dryness;
s5, dyeing:
s501, washing the dried slices obtained in the S404 with double-steaming water for 2 times, each time for 5min;
s502, dyeing in a dyeing tank filled with ammonia solution, wherein the dyed tissue is shown in figure 3: a, dyeing for 10min in dark, and washing with double distilled water for 2 times, each time for 5min;
the ammonia solution is prepared from ammonia water and double distilled water with the mass fraction of 25% in a volume ratio of 3:1;
the ammonia solution is prepared and used under the protection of the fume exhaust hood and the protective glove;
s503, washing the film in a 1% sodium thiosulfate solution for more than 15 min;
the preparation method of the 1% sodium thiosulfate solution comprises the steps of dissolving 3.14g of sodium thiosulfate pentahydrate in 200mL of double distilled water, and preparing and using under the protection of a smoke exhaust hood and protective gloves;
at this time, it should be noted that the washed tissue is basically white, the more yellow indicates that the washing is more disqualified, because the tissue is cracked after the tissue with yellow materials is baked and the yellow materials form impurities to influence the quality of the sheet after the sheet is sealed in the later period, as shown in fig. 3:B, the left two arrows show the disqualified tissue, and the right two arrows show the qualified tissue;
s504, after film washing, placing the film on a frame, horizontally placing the frame in double distilled water, enabling a tissue surface to face upwards, washing twice for 5 minutes each time, and if the film falling phenomenon occurs, placing the film into an oven for baking; at the moment, the patch is very easy to fall off due to water washing, but the patch is allowed to be carried out again at the moment, because the tissue is less rigid and more flexible than the tissue when being sliced, the tissue is not easy to damage, and if the tissue is uneven during the patch, the patch can be carried out again after the patch is fallen off at the moment; at this point, a microscopic examination can be performed, at which time the neuronal morphology can be seen substantially, as in fig. 3: c is shown as the specification;
s505, placing the slices in an oven with the temperature of 60 ℃ for baking for 2 hours in a dark place until the slices are completely dehydrated, so that the slices can be effectively prevented from falling; the baked tissue is as shown in fig. 3: d, if yes, referring to S503, and comparing whether the film is qualified;
FIG. 3 is a tissue staining protocol; wherein A is a tissue picture dyed by the ammonia solution; b: a tissue state comparison picture after washing with sodium thiosulfate pentahydrate is shown; c, the microscopic picture after water washing is shown; d, showing a tissue picture after baking;
s506, washing for 4 times, each time for 5min;
the step can also select whether to dye with 1% cresyl violet according to the need;
s6, dehydration and installation:
s601, dehydration and transparency: the stained slide obtained in S506 was placed in the following solutions in order, and dehydrated and transparent: 5min of ethanol aqueous solution with the mass fraction of 50%, 5min of ethanol aqueous solution with the mass fraction of 75%, 5min of ethanol aqueous solution with the mass fraction of 95%, 15min of absolute ethanol A, 25min of absolute ethanol A, 5min of dimethylbenzene I and 5min of dimethylbenzene II;
s602, sealing the film by using neutral resin, and storing the film in the dark until microscopic examination.
FIG. 4 is a graph showing the result of Golgi staining in this example, wherein A is a photograph of cerebral cortical neurons; b: a picture of hippocampal neurons is shown; c, a hypothalamic neuron picture is shown; d, displaying a purkinje neuron picture; e and F: a dendritic spine picture is shown.
As shown in FIG. 4, we observed clear cells stained with cerebral cortical neurons (A), hippocampal neurons (B) and hypothalamic neurons (C) at 50 μm using confocal laser photography; cerebellar purkinje neurons (D) were observed at 20 μm, and we observed clear dendritic spines at 20 μm (E) and 2 μm (F). The pictures are taken after the manufactured chips are stored for two months at room temperature in a dark place, and show that the dyed chips manufactured by the embodiment have long storage time, generate fewer impurities and do not influence analysis of dendritic spines.
Example 2
The method for applying the frozen section in the Golgi staining in the embodiment comprises the following steps:
s1, material selection: taking brain tissue of a C57BL/6J mouse, washing with double distilled water, cutting into blocks, and cutting into brain tissue blocks with the thickness of 0.5 cm;
s2, immersion liquid: soaking the brain tissue block obtained in the step S1 in a centrifuge tube filled with working solution for 6 hours, replacing the working solution, and keeping the soaked brain tissue block at room temperature in a dark place without replacing the working solution for 7 days;
the preparation method of the working solution comprises the following steps: vibrating and uniformly mixing the solution A, the solution B, the solution C and double distilled water according to the volume ratio of 5:5:4:10, reacting for 48 hours in the dark at room temperature, and filtering by filter paper to obtain working solution;
the solution A is prepared by dissolving 5g of potassium dichromate in 100mL of double distilled water;
the solution B is prepared by heating 5g of mercury chloride in a water bath at 90 ℃ and dissolving in 100mL of double distilled water;
the solution C is prepared by dissolving 5g of potassium sorbate in 100mL of double distilled water;
s3, soaking the cryoprotectant:
taking out the brain tissue blocks after the immersion liquid obtained in the step S2 by using a plastic clamp under the weak light environment of 100Lux, wiping the brain tissue blocks with absorbent paper, soaking the wiped brain tissue blocks in a cryoprotectant for 3 days, and replacing the cryoprotectant every 24 hours until the brain tissue blocks are sunk to obtain the brain tissue blocks after the cryoprotectant is soaked;
the cryoprotectant is prepared from the following raw materials in parts by weight: 20g of sucrose and 20mL of glycerol, and fixing the volume to 100mL by double distilled water;
s4, freezing and slicing:
s401, dropwise adding an OCT embedding agent into a precooled tray in a frozen microtome at the temperature of minus 25 ℃ to form an ice bottom, fixing the brain tissue block soaked with the cryoprotectant solution obtained in the S3 on the tray, dropwise adding the OCT embedding agent into the brain tissue block soaked with the cryoprotectant solution, and freezing for 40 minutes in the frozen microtome to obtain a frozen brain tissue block;
s402, slicing the brain tissue block frozen in S401 by using the frozen microtome, as shown in FIG. 1: a, after slicing, clamping the edge by forceps, and placing the edge into a 12-hole plate filled with 30% sucrose solution to spread the sheet to obtain a slice;
the 30% sucrose solution is prepared by dissolving 30g sucrose with 100mL double distilled water;
s403, sucking 30% sucrose solution on a glass slide hung with gelatin by using a Pasteur pipette to form a water area, moving the slice obtained in S402 on the glass slide by using a frozen slice blade, flattening the slice, discarding the redundant sucrose solution, and then adjusting the position of the slice by pulling a needle or moving tissues into a dish filled with 30% sucrose solution for pulling out the slice so that the slice is attached to the glass slide hung with gelatin, thereby obtaining attached slices;
s404, placing the pasted flakes obtained in the S403 in a dark place, and baking at 37 ℃ for 60min to obtain dried flakes;
s5, dyeing:
s501, washing the dried slices obtained in the S404 with double-steaming water for 2 times, each time for 5min;
s502, dyeing in a dyeing tank filled with ammonia solution, wherein the dyed tissue is shown in figure 3: a, dyeing for 10min in dark, and washing with double distilled water for 2 times, each time for 5min;
the ammonia solution is prepared from 28% ammonia water and double distilled water in a volume ratio of 3:1;
s503, washing the film in a 1% sodium thiosulfate solution for more than 15 min;
s504, after film washing, placing the film on a frame, horizontally placing the frame in double distilled water, and washing twice for 5min each time with the organized surface upwards;
s505, placing the flakes in an oven with the temperature of 60 ℃ and baking for 3 hours in a dark place;
s506, washing for 4 times, each time for 5min;
s6, dehydration and installation:
s601, dehydration and transparency: the stained slide obtained in S506 was placed in the following solutions in order, and dehydrated and transparent: 5min of ethanol aqueous solution with the mass fraction of 50%, 5min of ethanol aqueous solution with the mass fraction of 75%, 5min of ethanol aqueous solution with the mass fraction of 95%, 15min of absolute ethanol A, 25min of absolute ethanol A, 5min of dimethylbenzene I and 5min of dimethylbenzene II;
s602, sealing the film by using neutral resin, and storing the film in the dark until microscopic examination.
The above description is only of the preferred embodiments of the present invention, and is not intended to limit the present invention. Any simple modification, variation and equivalent variation of the above embodiments according to the technical substance of the invention still fall within the scope of the technical solution of the invention.

Claims (2)

1. A method for applying frozen sections to golgi staining, comprising:
s1, material selection: taking brain tissue of a C57BL/6J mouse, washing with double distilled water, cutting into blocks, and cutting into brain tissue blocks with the thickness of 0.5 cm;
s2, immersion liquid: soaking the brain tissue block obtained in the step S1 in a centrifuge tube filled with working solution for 6-24 hours, replacing the working solution, and keeping the soaked brain tissue block at room temperature in a dark place without replacing the working solution after the soaking for 7-14 days;
the preparation method of the working solution comprises the following steps: vibrating and uniformly mixing the solution A, the solution B, the solution C and double distilled water according to the volume ratio of 5:5:4:10, reacting for 24-48 hours in the dark at room temperature, and filtering by a 0.22 mu mm filter or filter paper to obtain working solution;
the solution A is prepared by dissolving 5g of potassium dichromate in 100mL of double distilled water;
the solution B is prepared by heating 5g of mercury chloride in a water bath at 90 ℃ and dissolving in 100mL of double distilled water;
the solution C is prepared by dissolving 5g of potassium sorbate in 100mL of double distilled water;
s3, soaking the cryoprotectant:
taking out the brain tissue blocks after immersion, which are obtained in the step S2, by using a plastic clamp under the illumination environment of 100Lux, wiping the brain tissue blocks with absorbent paper, then putting the wiped brain tissue blocks into a cryoprotectant for soaking for 2-3 days, and replacing the cryoprotectant every 24 hours until the brain tissue blocks are submerged, thus obtaining the brain tissue blocks after soaking the cryoprotectant;
the cryoprotectant is prepared from the following raw materials in parts by weight: 20g of sucrose and 15-20 mL of glycerol, and the volume is fixed to 100mL by double distilled water;
s4, freezing and slicing:
s401, dropwise adding an OCT embedding agent into a precooled tray in a frozen microtome at the temperature of minus 25 ℃ to minus 20 ℃ to form an ice bottom, fixing the brain tissue block soaked with the cryoprotectant liquid obtained in the S3 on the tray, dropwise adding the OCT embedding agent into the brain tissue block soaked with the cryoprotectant liquid, and freezing in the frozen microtome for more than 30 minutes to obtain a frozen brain tissue block;
s402, slicing the brain tissue block frozen in the S401 by using the frozen microtome, clamping the edge by using forceps after slicing, and placing the frozen brain tissue block into a pore plate filled with 30% sucrose solution for expanding to obtain slices;
the 30% sucrose solution is prepared by dissolving 30g sucrose with 100mL double distilled water;
s403, sucking 30% sucrose solution on a glass slide hung with gelatin by using a Pasteur pipette to form a water area, moving the slice obtained in S402 on the glass slide by using a frozen slice blade, flattening the slice, discarding the redundant sucrose solution, and then adjusting the position of the slice by pulling a needle or moving tissues into a dish filled with 30% sucrose solution for pulling out the slice so that the slice is attached to the glass slide hung with gelatin, thereby obtaining attached slices;
s404, placing the stuck pieces obtained in the S403 in a dark place, standing overnight at room temperature under a ventilation condition or baking at 37 ℃ for 30-60 min to obtain dried pieces;
s5, dyeing:
s501, washing the dried slices obtained in the S404 with double-steaming water for 2 times, each time for 5min;
s502, dyeing in a dyeing tank filled with ammonia solution for 10min in a dark place, and washing with double-distilled water for 2 times, wherein each time lasts for 5min;
the ammonia solution is prepared from ammonia water and double distilled water with the mass fraction of 25% -28% in a volume ratio of 3:1;
s503, washing the film in a 1% sodium thiosulfate solution for more than 15 min;
s504, after film washing, placing the film on a frame, horizontally placing the frame in double distilled water, and washing twice for 5min each time with the organized surface upwards;
s505, placing the flakes in an oven with the temperature of 60 ℃ and baking for 2-3 hours in a dark place;
s506, washing for 4 times, each time for 5min;
s6, dehydration and installation:
s601, dehydration and transparency: the stained slide obtained in S506 was placed in the following solutions in order, and dehydrated and transparent: 5min of ethanol aqueous solution with the mass fraction of 50%, 5min of ethanol aqueous solution with the mass fraction of 75%, 5min of ethanol aqueous solution with the mass fraction of 95%, 15min of absolute ethanol A, 25min of absolute ethanol A, 5min of dimethylbenzene I and 5min of dimethylbenzene II;
s602, sealing the film by using neutral resin, and storing the film in the dark until microscopic examination.
2. A method of using frozen sections in golgi staining according to claim 1, wherein the well plate in S402 is a 6-well plate or a 12-well plate.
CN202310780327.4A 2023-06-29 2023-06-29 Application method of frozen section in Golgi staining Pending CN116718452A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310780327.4A CN116718452A (en) 2023-06-29 2023-06-29 Application method of frozen section in Golgi staining

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310780327.4A CN116718452A (en) 2023-06-29 2023-06-29 Application method of frozen section in Golgi staining

Publications (1)

Publication Number Publication Date
CN116718452A true CN116718452A (en) 2023-09-08

Family

ID=87871387

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310780327.4A Pending CN116718452A (en) 2023-06-29 2023-06-29 Application method of frozen section in Golgi staining

Country Status (1)

Country Link
CN (1) CN116718452A (en)

Similar Documents

Publication Publication Date Title
CN105973673B (en) A kind of paraffin section method of eucalyptus tissue
CN109855936B (en) Preparation method of plant tissue section with complete tissue and favorable microscopic observation
CN102967493B (en) Rapid paraffin sectioning method for plant tissue
CN107702959A (en) A kind of paraffin section method for vegetable material serial dehydration
CN110132673B (en) Method for preparing needle mushroom fruiting body tissue slices
CN111257089B (en) Optimized manufacturing method of paraffin sections of different flower tissues of butterfly orchid
CN109374373A (en) A kind of Apple paraffin section fast method for preparing
CN106501043B (en) A kind of paraffin section method of effective observation oil palm gynoecium anatomical structure
CN103257055A (en) Preparation method of hyriopsis cumingii pallium tissue slice
CN105547793B (en) A kind of corn mature seed farinaceous albumen nail polish aids in whole slices preparation method
CN107655731B (en) Paraffin section method for leaf buds and flower buds of larger explants of carnation
CN109060468A (en) A kind of preparation method of the paraffin section of pear flower tissue
CN116718452A (en) Application method of frozen section in Golgi staining
CN110926907A (en) Method suitable for making equine animal ovary paraffin section
CN107202720B (en) A kind of paraffin section method of pomegranate seed
Sylvester et al. Light microscopy I: dissection and microtechnique
JP3723204B1 (en) Impermeable tissue quick fixative
CN111896353A (en) Small tissue block dehydration method
CN115683697A (en) Method for slicing plant phloem tissue
CN109859304B (en) Three-dimensional printing technology for establishing three-dimensional structure digital model outside cornea limbal tissue
Engen et al. N–butyl Methacrylate and Paraffin as an Embedding Medium for Light Microscopy
Abdelbar Histological analysis of the developmental stages of direct somatic embryogenesis induced from in vitro leaf explants of date palm
CN106840809A (en) A kind of flat pathology bakes piece machine and its water logging device
CN113984483A (en) Staining method for frozen section cover glass paster
CN112798391A (en) Method for preparing bulbil paraffin embedding slice

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination