CN110426264B - Method for staining fat cells and application thereof - Google Patents
Method for staining fat cells and application thereof Download PDFInfo
- Publication number
- CN110426264B CN110426264B CN201910797148.5A CN201910797148A CN110426264B CN 110426264 B CN110426264 B CN 110426264B CN 201910797148 A CN201910797148 A CN 201910797148A CN 110426264 B CN110426264 B CN 110426264B
- Authority
- CN
- China
- Prior art keywords
- adipose tissue
- frozen sections
- staining
- tritonx
- dmso
- 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.)
- Active
Links
- 238000010186 staining Methods 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims abstract description 37
- 210000001789 adipocyte Anatomy 0.000 title claims description 27
- 210000000577 adipose tissue Anatomy 0.000 claims abstract description 109
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 106
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 claims abstract description 39
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims abstract description 36
- NPGIHFRTRXVWOY-UHFFFAOYSA-N Oil red O Chemical compound Cc1ccc(C)c(c1)N=Nc1cc(C)c(cc1C)N=Nc1c(O)ccc2ccccc12 NPGIHFRTRXVWOY-UHFFFAOYSA-N 0.000 claims abstract description 25
- 210000004292 cytoskeleton Anatomy 0.000 claims abstract description 16
- 238000007447 staining method Methods 0.000 claims abstract description 16
- 238000012545 processing Methods 0.000 claims abstract description 10
- 239000003153 chemical reaction reagent Substances 0.000 claims description 33
- 239000008363 phosphate buffer Substances 0.000 claims description 30
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 29
- 239000011521 glass Substances 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
- 238000005406 washing Methods 0.000 claims description 22
- 239000012153 distilled water Substances 0.000 claims description 20
- 239000000872 buffer Substances 0.000 claims description 17
- 239000007853 buffer solution Substances 0.000 claims description 17
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 15
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 15
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 10
- 238000004042 decolorization Methods 0.000 claims description 10
- 239000008055 phosphate buffer solution Substances 0.000 claims description 10
- 239000006059 cover glass Substances 0.000 claims description 9
- 150000002632 lipids Chemical class 0.000 claims description 9
- GNFTZDOKVXKIBK-UHFFFAOYSA-N 3-(2-methoxyethoxy)benzohydrazide Chemical compound COCCOC1=CC=CC(C(=O)NN)=C1 GNFTZDOKVXKIBK-UHFFFAOYSA-N 0.000 claims description 8
- FGUUSXIOTUKUDN-IBGZPJMESA-N C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 Chemical compound C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 FGUUSXIOTUKUDN-IBGZPJMESA-N 0.000 claims description 8
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 5
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 claims description 5
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 5
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 5
- 238000004043 dyeing Methods 0.000 description 12
- 238000001000 micrograph Methods 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 8
- 239000000463 material Substances 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 6
- 239000000834 fixative Substances 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 238000000386 microscopy Methods 0.000 description 4
- 238000012449 Kunming mouse Methods 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 101100102907 Mus musculus Wdtc1 gene Proteins 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- SGHZXLIDFTYFHQ-UHFFFAOYSA-L Brilliant Blue Chemical compound [Na+].[Na+].C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C(=CC=CC=2)S([O-])(=O)=O)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 SGHZXLIDFTYFHQ-UHFFFAOYSA-L 0.000 description 1
- 238000011537 Coomassie blue staining Methods 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000037237 body shape Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000003436 cytoskeletal effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000010827 pathological analysis Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000011546 protein dye Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
Images
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
-
- 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
- G01N1/30—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
- G01N1/28—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
- G01N1/31—Apparatus therefor
- G01N1/312—Apparatus therefor for samples mounted on planar substrates
-
- 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
- G01N1/34—Purifying; Cleaning
-
- 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
-
- 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
- G01N2001/302—Stain compositions
-
- 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
- G01N2001/305—Fixative compositions
Abstract
The invention discloses an adipose tissue staining method, which comprises the following steps: a. processing the frozen sections of the adipose tissues by using TritonX-100; b. removing TritonX-100; c. fixing the slices by glutaraldehyde; d. staining the sections with oil red O; e. decoloring the slices; f. treating the slices with DMSO; g. removing the DMSO; h. sections were stained with coomassie brilliant blue R250; i. and (6) decoloring. For staining the cytoskeleton. The invention discloses another adipose tissue staining method, which comprises the following steps: a. treating frozen sections of adipose tissue with DMSO; b. removing the DMSO; c. processing the slices by using TritonX-100; d. removing TritonX-100; e. fixing the slices by glutaraldehyde; f. removing glutaraldehyde; g. sections were stained with coomassie brilliant blue R250; h. and (6) decoloring. The invention also discloses two kits and applications of the two methods.
Description
Technical Field
The invention belongs to the technical field of biology, relates to a staining method of fat cells and application thereof, and particularly relates to a method capable of simultaneously representing fat droplets and cytoskeleton in the fat cells and application thereof.
Background
The oil red O fat staining method is a method for staining fat in tissues in order to show the fat in the tissues in daily pathological diagnosis and scientific research work by adopting oil red O, wherein the oil red O is a fat-soluble dye which can be highly dissolved in the fat and can specifically stain neutral fat such as triglyceride and the like in the tissues.
The method is used for staining fat cells, can stain fat droplets in the fat cells, but is too narrow in application and limited in exploitable range, and cannot be used for staining cytoskeletons.
The common method for displaying cytoskeleton is a Coomassie brilliant blue staining method, has the characteristics of simplicity and feasibility, cannot distinguish the composition of the framework protein, and can only be used for the research of the framework morphology and integrity.
The cell is treated with detergent TritonX-100 to dissolve membrane lipid, and combines with most of hydrophobic region of non-skeleton protein to dissolve it, and the protein of the remaining cytoskeletal system is not dissolved, and then stained with protein dye Coomassie brilliant blue to display the skeleton structure.
The method is not suitable for staining the fat cell skeleton, because fat cells are rich in oil and are not easy to stain.
Because the existing technology can not stain fat cell lipid drops and cytoskeleton well at the same time, there are only reports on the research on the relationship between fat cell lipid drops and cytoskeleton.
Disclosure of Invention
In order to solve the defects of the prior art, the invention designs a mixed staining method by utilizing the characteristics of fat droplets and cytoskeleton of fat cells. The method can be used for researching the relation between lipid droplets and cytoskeleton, has simple dyeing steps, convenient operation and good dyeing effect, and can be completely used for scientific research on fat cells.
The invention utilizes the combination and innovation of two staining methods, can clearly show the shape distribution of fat droplets and a skeleton of the fat cells, and can be used for the research on the fat cells.
More specifically, the present invention provides in a first aspect a method for staining adipose tissue, whereby
The dyeing method comprises the following steps:
a. processing the frozen sections of the adipose tissues by using TritonX-100;
b. removing the TritonX-100;
c. fixing the frozen sections of adipose tissue with glutaraldehyde;
d. staining the adipose tissue frozen sections with oil red O;
e. decolorizing the frozen section of the adipose tissue;
f. treating the frozen sections of adipose tissue with DMSO;
g. removing the DMSO;
h. staining the frozen sections of adipose tissue with coomassie brilliant blue R250;
i. and (6) decoloring.
In some embodiments, prior to step a, the frozen sections of adipose tissue are rinsed with phosphate buffer;
in some embodiments, the frozen slice of adipose tissue has a thickness of 30-40 μm;
in some embodiments, in step a, frozen sections of adipose tissue are treated with 0.8-1.2% TritonX-100 for 15-25 min;
in some embodiments, in step b, the triton x-100 is removed with M buffer;
in some embodiments, in step b, wash 1-3 times with M buffer, 3-6min each time;
in some embodiments, in step c, the frozen sections of adipose tissue are fixed with 2.5-3.5% glutaraldehyde for 20-40 min;
in some embodiments, in step d, the frozen sections of adipose tissue are stained with oil red O for 8-12 min;
in some embodiments, in step e, the frozen sections of adipose tissue are destained with isopropanol;
in some embodiments, the isopropanol concentration is 55-65% and the treatment time is from rinsing to washing off bloom;
in some embodiments, in step f, the frozen sections of adipose tissue are treated with 45-55%% DMSO for 5-10 minutes;
in some embodiments, in step g, DMSO is removed by rinsing with phosphate buffer;
in some embodiments, in step h, the frozen section of adipose tissue is stained with 0.15-0.25% coomassie brilliant blue R250 for 25-35 min;
in some embodiments, in step i, the decolorization is washed with distilled water.
In some embodiments, prior to step a, the frozen sections of adipose tissue are rinsed three times, one minute each, with phosphate buffer;
in some embodiments, the frozen slice of adipose tissue is 35 μm thick;
in some embodiments, in step a, frozen sections of adipose tissue are treated with 1% triton x-100 for 20 min;
in some embodiments, in step b, the triton x-100 is removed three times for 5min each with M buffer;
in some embodiments, in step b, three washes with M buffer for 5min each;
in some embodiments, in step c, the frozen sections of adipose tissue are fixed with 3% glutaraldehyde for 30 min;
in some embodiments, in step d, the frozen section of adipose tissue is stained with oil red O for 10 min;
in some embodiments, in step f, the frozen sections of adipose tissue are treated with 50% DMSO for 8 minutes;
in some embodiments, in step g, DMSO is removed by rinsing once with phosphate buffer;
in some embodiments, in step h, the frozen section of adipose tissue is stained with 0.2% coomassie brilliant blue R250 for 30 min;
in some embodiments, in step i, the decolorization is performed by washing with distilled water twice;
preferably, after step i, the sample is placed on a slide and coverslipped.
In a second aspect, the present invention provides a kit for adipose tissue staining, the kit comprising:
a reagent bottle for containing or containing a phosphate buffer solution;
the reagent bottle is used for containing or containing TritonX-100;
the reagent bottle is used for containing or containing M buffer solution;
a reagent bottle for containing or containing glutaraldehyde;
the reagent bottle is used for containing or containing oil red O;
a reagent bottle for containing or containing isopropanol;
a reagent bottle for containing or containing DMSO;
a reagent bottle for containing or containing Coomassie brilliant blue R250;
in some embodiments, the kit is used in the adipose tissue staining method according to the first aspect of the invention;
in some embodiments, the kit further comprises:
the reagent bottle is used for containing or containing distilled water;
in some embodiments, the kit further comprises:
a glass slide;
in some embodiments, the kit further comprises:
and (5) covering with a glass slide.
In a third aspect, the present invention provides a method for staining adipose tissue, comprising the steps of:
a. treating frozen sections of adipose tissue with DMSO;
b. removing the DMSO;
c. processing the frozen sections of the adipose tissues by using TritonX-100;
d. removing the TritonX-100;
e. fixing the frozen sections of adipose tissue with glutaraldehyde;
f. removing glutaraldehyde;
g. staining the frozen sections of adipose tissue with coomassie brilliant blue R250;
h. and (6) decoloring.
In some embodiments, prior to step a, the frozen sections of adipose tissue are rinsed with phosphate buffer;
in some embodiments, the frozen slice of adipose tissue has a thickness of 30-40 μm;
in some embodiments, in step a, the frozen sections of adipose tissue are treated with 45-55% DMSO for 5-10 minutes;
in some embodiments, in step c, frozen sections of adipose tissue are treated with 0.8-1.2% TritonX-100 for 15-25 min;
in some embodiments, in step d, the triton x-100 is removed with M buffer;
in some embodiments, in step d, wash 1-3 times with M buffer, 3-6min each time;
in some embodiments, in step e, the frozen sections of adipose tissue are fixed with 2.5-3.5% glutaraldehyde for 20-40 min;
in some embodiments, in step f, glutaraldehyde is removed by rinsing with phosphate buffer;
in some embodiments, in step g, the frozen section of adipose tissue is stained with 0.15-0.25% coomassie brilliant blue R250 for 25-35 min;
in some embodiments, in step h, the decolorization is washed with distilled water.
In some embodiments, prior to step a, the frozen sections of adipose tissue are rinsed three times, one minute each, with phosphate buffer;
in some embodiments, the frozen slice of adipose tissue is 35 μm thick;
in some embodiments, in step a, the frozen sections of adipose tissue are treated with 50% DMSO for 8 minutes;
in some embodiments, in step c, frozen sections of adipose tissue are treated with 1% triton x-100 for 20 min;
in some embodiments, in step d, the triton x-100 is removed three times for 5min each with M buffer;
in some embodiments, in step e, the frozen sections of adipose tissue are fixed with 3% glutaraldehyde for 30 min;
in some embodiments, in step f, three rinses with phosphate buffer remove glutaraldehyde;
in some embodiments, in step g, the frozen section of adipose tissue is stained with 0.2% coomassie brilliant blue R250 for 30 min;
in some embodiments, in step h, the decolorization is washed twice with distilled water;
in some embodiments, after step h, the sample is placed on a slide and coverslipped.
In a fourth aspect, the present invention provides a kit for adipose tissue staining, the kit comprising:
a reagent bottle for containing or containing a phosphate buffer solution;
a reagent bottle for containing or containing DMSO;
the reagent bottle is used for containing or containing TritonX-100;
the reagent bottle is used for containing or containing M buffer solution;
a reagent bottle for containing or containing glutaraldehyde;
a reagent bottle for containing or containing Coomassie brilliant blue R250;
in some embodiments, the kit is used in the adipose tissue staining method according to the third aspect of the present invention;
in some embodiments, the kit further comprises:
the reagent bottle is used for containing or containing distilled water;
in some embodiments, the kit further comprises:
a glass slide;
in some embodiments, the kit further comprises:
and (5) covering with a glass slide.
In a fifth aspect the invention provides a method according to the first aspect of the invention, or a kit according to the second aspect of the invention, for use or use in simultaneously characterising fat cell lipid droplets and a cytoskeleton or the relationship between said fat cell lipid droplets and said cytoskeleton.
In a sixth aspect, the invention provides a method according to the third aspect of the invention, or the use or use of a kit according to the fourth aspect of the invention, for characterising the cytoskeleton of adipocytes.
Drawings
FIG. 1 is a micrograph of a stained specimen at X100 of example 1 of the present invention.
FIG. 2 is a micrograph of a stained specimen at X20 of example 2 of the present invention.
FIG. 3 is a micrograph of a stain at X20 of example 3 of the present invention.
FIG. 4 is a micrograph of a stain at X20 of example 4 of the present invention.
FIG. 5 is a photomicrograph of a stain at X20 of example 5 of the present invention.
FIG. 6 is a micrograph of a stain at X40 of example 6 of the present invention.
FIG. 7 is a micrograph of a stain at X20 of example 7 of the present invention.
FIG. 8 is a photomicrograph of a stain at 100 of example 7 of the present invention.
FIG. 9 is a photomicrograph of a stain at 100 of example 8 of the present invention.
FIG. 10 is a photomicrograph of a stain at X40 of example 9 of the present invention.
Detailed Description
In order to better explain the technical scheme of the invention, the following detailed description of the embodiment of the invention is combined with the accompanying drawings. The following examples are intended to further illustrate the invention but should not be construed as being limitations or restrictive thereon. Unless otherwise specified, technical features used in the embodiments may be replaced with other technical features known in the art having equivalent or similar functions or effects without departing from the inventive concept.
Materials and methods
1 materials of the experiment
The instrument comprises the following steps: a cryomicrotome (CM 1900);
animal materials: the Kunming mouse for the experiment is provided by the Anhui labour engineering technical research center of anti-aging Chinese herbal medicine (Anhui Fuyang); filtering paper; a glass slide; and (3) O.C.T.
Reagent: 0.2% Coomassie Brilliant blue R250 dye liquor (0.2% (W/V) Coomassie Brilliant blue R-250, 25% (V/V) isopropanol, 10% (V/V) glacial acetic acid); 0.5% oil red O dyeingLiquid (oil red O powder 0.5g, adding isopropanol 100ml, water bath at 60 deg.C to dissolve completely, making into stock solution, adding distilled water 4ml into stock solution 6ml when using, filtering, and using); phosphate buffered saline (PBS, pH7.5); 50% aqueous dimethyl sulfoxide (DMSO); 1% TritonX-100; m buffer (imidazole 50 mmol/L3.40 g, KCl 50 mmol/L3.73 g, MgCl2·6H2O 0.5mmol/L 0.1g，EGTA 1.0mmol/L 0.38g，EDTA·2H2O0.1 mmol/L0.04g, B-mercaptoethanol 1.0 mmol/L70 ul, glycerol 4.0 mmol/L294.8 ul, adding water to constant volume of 1L, and adjusting pH to 7.2); 3% glutaraldehyde solution in water; 60% isopropanol.
2 obtaining of test materials
Dissecting Kunming mice with normal body shape, and taking the inguinal adipose tissue of the Kunming mice.
3 preparing tablets
Frozen sections of mouse adipose tissue were made in a conventional manner using a cryomicrotome (leica CM 1900).
Because the frozen section of the adipose tissue is not easy to operate, the cell shape is easy to be influenced by temperature, and the section stripping phenomenon is easy to occur in the dyeing process, the invention adopts the technical scheme that the used glass slide is placed in the frozen section machine in advance to reduce the influence of temperature on the adipose tissue section, the section with good cell shape can be obtained by using a thicker frozen adipose tissue section (the section thickness is 35 mu m), the section cannot be baked due to the fact that the adipose cells are rich in grease, and the tissue section is pasted by placing for 1-2 min at room temperature.
The foregoing materials and methods are used in the following examples, where not indicated, the same process steps and parameters are used. The examples, as well as the foregoing materials and methods, are not mentioned as being conventional in the art and are within the common general knowledge.
Example 1: improved fat cell section Coomassie brilliant blue R250 staining method
1. Frozen adipose tissue sections (section thickness 35 μm) were rinsed three times in phosphate buffer for 1min each, and treated with 50% DMSO for 8 min.
2. DMSO was removed and 2ml of 1% TritonX-100 was added for 20 min.
3. TritonX-100 was removed and washed three times with M buffer for 5min each.
4. Fixation with 3% glutaraldehyde for 30 min.
5. The fixative was removed and gently washed three times each for 3min with phosphate buffer.
6. Stain with 0.2% Coomassie Brilliant blue R250 for 30 min.
7. The sample was washed twice with distilled water, placed on a glass slide, and subjected to microscopic examination with a cover glass.
Micrograph of staining at x 100 is shown in figure 1. It can be seen from FIG. 1 that the method of example 1 can well visualize cytoskeleton in adipocytes.
As can be seen from FIG. 1, the improved Coomassie brilliant blue R250 staining method of example 1 solves the problem that the conventional staining method cannot stain the adipose cell skeletons, and can present clear adipose cell skeletons.
Example 2: coomassie brilliant blue staining method
A frozen section of the mouse adipose tissue is prepared by using a freezing microtome (leica CM1900), the section is directly placed on a glass slide which is not subjected to cooling treatment, and the glass slide is placed at room temperature for 1-2 min to enable the tissue section to be pasted.
And (3) dyeing:
1. frozen adipose tissue sections (section thickness 35 μm) were rinsed three times in phosphate buffer, one minute each, with 50% DMSO for 8 minutes;
2. removing DMSO, adding 2mL of 1% TritonX-100, and treating for 20 min;
3. removing TritonX-100, washing with M buffer solution for three times, each time for 5 min;
4. fixing with 3% glutaraldehyde for 30 min;
5. removing the fixative, and gently washing with phosphate buffer solution for three times, each for 3 min;
6. staining with 0.2% Coomassie Brilliant blue R250 for 30 min;
7. the sample was washed twice with distilled water, placed on a glass slide, and subjected to microscopic examination with a cover glass.
The results of the x 20 microscopy are shown in figure 2.
As can be seen from fig. 2, the use of the glass slide without temperature reduction treatment resulted in the damage of cell morphology, and thus, the use of the glass slide with temperature reduction treatment was more effective and easier to preserve the intact cell morphology.
The difference between example 1 and example 2 is that example 2 uses a glass slide which has not been subjected to temperature reduction treatment, whereas example 1 uses a glass slide which has been subjected to temperature reduction treatment as in the other examples. Therefore, the color development picture can be more complete and the effect is clearer by using the glass slide subjected to temperature reduction treatment.
Example 3: coomassie brilliant blue staining
The experimental steps are as follows:
1. rinsing frozen adipose tissue slices (slice thickness 35 μm) in phosphate buffer for three times, each time for 1 min;
2. removing the phosphate buffer, adding 2mL of 1% TritonX-100, and treating for 20 min;
3. removing TritonX-100, washing with M buffer solution for three times, each time for 5 min;
4. fixing with 3% glutaraldehyde for 30 min;
5. removing the fixative, and gently washing with phosphate buffer solution for three times, each for 3 min;
6. staining with 0.2% Coomassie Brilliant blue R250 for 30 min;
7. the sample was washed twice with distilled water, placed on a glass slide, and subjected to microscopic examination with a cover glass.
The results of the x 20 microscopy are shown in figure 3.
As can be seen from a comparison of fig. 1 and fig. 3, no DMSO treatment resulted in no staining of the intracellular scaffold, indicating that DMSO treatment was able to promote coomassie brilliant blue R250 staining.
Example 4: coomassie brilliant blue staining
1. Frozen adipose tissue sections (section thickness 35 μm) were rinsed three times in phosphate buffer, 1min each time, and treated with 50% DMSO for 15 min;
2. removing the phosphate buffer, adding 2mL of 1% TritonX-100, and treating for 20 min;
3. removing TritonX-100, washing with M buffer solution for three times, each time for 5 min;
4. fixing with 3% glutaraldehyde for 30 min;
5. removing the fixative, and gently washing with phosphate buffer solution for three times, each for 3 min;
6. staining with 0.2% Coomassie Brilliant blue R250 for 30 min;
7. the sample was washed twice with distilled water, placed on a glass slide, and subjected to microscopic examination with a cover glass.
The results of the x 20 microscopy are shown in fig. 4.
As can be seen in FIG. 4, the DMSO treatment time in the Coomassie blue staining was too long, resulting in a disruption of cell morphology. The DMSO treatment time cannot be too long.
Example 5: oil red O dyeing
1. Frozen sections of adipose tissue (slice thickness 35 μm) were rinsed three times for 1min each with phosphate buffer.
2.3% glutaraldehyde for 30 min.
3. Oil red O staining for 10 min.
Decolorized with 4.60% isopropanol and examined under a microscope.
See figure 5 for a micrograph of the staining at x 20. It can be seen from FIG. 5 that the method of example 5 can well visualize fat droplets in adipocytes.
Example 6: oil red O dyeing
The experimental process comprises the following steps:
1. frozen sections of adipose tissues (the thickness of the sections is 35 mu m) are rinsed three times for 1min each time by using a phosphate buffer;
fixing with 2.3% glutaraldehyde for 30 min;
3. treating with chemical pure DMSO for 8 min;
4. dyeing for 10min by using oil red O;
5.60% isopropanol was decolorized and examined under a microscope.
See figure 6 for a micrograph of the staining at x 40. As can be seen from FIG. 6, the effect of treatment with chemically pure DMSO in the oil-Red-O staining was that the cell morphology was destroyed in the method of example 6.
Example 7: oil red O and Coomassie brilliant blue R250 mixed dyeing method
1. Frozen sections of adipose tissues (the thickness of the sections is 35 mu m) are rinsed three times for 1min each time by using a phosphate buffer;
2. 2ml of 1% TritonX-100 was added and the mixture was treated for 20 min.
3. TritonX-100 was removed and washed three times with M buffer for 5min each.
4. Fixation with 3% glutaraldehyde for 30 min.
5. Oil red O staining for 10 min.
6.60% isopropanol.
7.50% DMSO for 8 min.
8. The DMSO was removed and rinsed once with phosphate buffer.
9. Stain with 0.2% Coomassie Brilliant blue R250 for 30 min.
10. And (4) washing with distilled water for two times for decolorization, placing the sample on a glass slide, adding a cover glass, and performing microscopic examination.
The micrographs of the staining at x 20 and x 100 are shown in fig. 7 and fig. 8. From fig. 7 and 8, it can be seen that the oil red O and coomassie brilliant blue R250 mixed staining method of example 7 can clearly show the distribution and morphology of lipid droplets and cytoskeleton in adipocytes, and can be used for scientific research, and the method steps are simple and effective, and have innovativeness and practicability.
Example 8: oil red O and Coomassie brilliant blue R250 mixed dyeing method
The experimental process comprises the following steps:
1. frozen sections of adipose tissues (the thickness of the sections is 35 mu m) are rinsed three times for 1min each time by using a phosphate buffer;
fixing with 2.3% glutaraldehyde for 30 min;
3. dyeing for 10min by using oil red O;
4.60% isopropyl alcohol decolorization;
4. rinsing with phosphate buffer solution for 1 min;
5. removing the phosphate buffer, adding 2mL of 1% TritonX-100, and treating for 20 min;
6. removing TritonX-100, washing with M buffer solution for three times, each time for 5 min;
7. fixing with 3% glutaraldehyde for 30 min;
8. removing the fixative, and gently washing with phosphate buffer solution for three times, each for 3 min;
9. staining with 0.2% Coomassie Brilliant blue R250 for 10 min;
10. the sample was washed twice with distilled water, placed on a glass slide, and subjected to microscopic examination with a cover glass.
See figure 9 for a micrograph of the staining at x 100.
As can be seen from FIG. 9, most cells of this example remain only in an empty shell.
Example 9:
1. frozen sections of adipose tissues (the thickness of the sections is 35 mu m) are rinsed three times for 1min each time by using a phosphate buffer;
2. adding 2ml of 1% TritonX-100, and processing for 20 min;
3. removing TritonX-100, washing with M buffer solution for three times, each time for 5 min;
4. fixing with 3% glutaraldehyde for 30 min;
5.50% DMSO for 8 min;
6. removing DMSO, rinsing with phosphate buffer solution once, staining with 0.2% Coomassie brilliant blue R250 for 30min, and decolorizing with distilled water;
7. dyeing for 10min by using oil red O;
decolorizing with 8.60% isopropanol, placing the sample on a glass slide, adding a cover glass, and performing microscopic examination.
The x 40 microscopy results are shown in figure 10.
The results of the first coomassie red O and then the second horse brilliant blue staining in fig. 7-8 are better than the first coomassie brilliant blue and then the second oil red O staining in fig. 10.
The above embodiments are only used for further illustration of the present invention, and are not intended to limit the scope of the present invention, and all equivalent changes made based on the concept of the present invention and obvious modifications of various technical solutions of the present invention fall within the scope of the present invention.
Claims (10)
1. A method of adipose tissue staining, the staining method comprising the steps of:
a. processing the frozen sections of the adipose tissues by using TritonX-100;
b. removing the TritonX-100;
c. fixing the frozen sections of adipose tissue with glutaraldehyde;
d. staining the adipose tissue frozen sections with oil red O;
e. decolorizing the frozen section of the adipose tissue;
f. treating the frozen sections of adipose tissue with DMSO;
g. removing the DMSO;
h. staining the frozen sections of adipose tissue with coomassie brilliant blue R250;
i. and (6) decoloring.
2. The method of adipose tissue staining of claim 1, wherein:
rinsing the frozen sections of adipose tissue with phosphate buffer prior to step a;
the thickness of the frozen section of the adipose tissue is 30-40 mu m;
in the step a, processing frozen sections of the adipose tissues by 0.8-1.2% TritonX-100 for 15-25 min;
in step b, the TritonX-100 is removed with M buffer, wherein M buffer comprises imidazole 50mmol/L, KCl 50mmol/L, MgCl2·6H2O 0.5mmol/L、EGTA 1.0mmol/L、EDTA·2H2O0.1 mmol/L, B-mercaptoethanol 1.0mmol/L, glycerol 4.0mmol/L, and M buffer solution pH 7.2;
in step b, washing with M buffer solution for 1-3 times, each time for 3-6 min;
in step c, fixing the frozen sections of the adipose tissues with 2.5-3.5% glutaraldehyde for 20-40 min;
in step d, staining the frozen sections of the adipose tissues with oil red O for 8-12 min;
in step e, decolorizing the frozen sections of adipose tissue with isopropanol;
the concentration of the isopropanol is 55-65%, and the treatment time is washing to wash away the floating color;
in step f, treating the frozen sections of adipose tissue with 45-55% DMSO for 5-10 minutes;
in step g, DMSO was removed by rinsing with phosphate buffer;
in step h, staining the frozen sections of adipose tissue with 0.15-0.25% Coomassie Brilliant blue R250 for 25-35 min;
in step i, washing with distilled water for decolorization.
3. The method of adipose tissue staining of claim 2, wherein:
rinsing the frozen sections of adipose tissue three times for one minute each with phosphate buffer prior to step a;
the thickness of the frozen section of the adipose tissue is 35 mu m;
in the step a, processing frozen sections of the adipose tissues by using 1% TritonX-100 for 20 min;
in step b, removing the TritonX-100 three times with M buffer, each time for 5 min;
in step b, washing with M buffer three times for 5min each time;
in step c, the frozen sections of adipose tissue were fixed with 3% glutaraldehyde for 30 min;
in step d, staining the frozen sections of adipose tissue with oil red O for 10 min;
in step f, treating the frozen sections of adipose tissue with 50% DMSO for 8 minutes;
in step g, DMSO was removed by rinsing once with phosphate buffer;
in step h, the frozen sections of adipose tissue were stained with 0.2% coomassie brilliant blue R250 for 30 min;
in step i, washing with distilled water twice for decolorization;
after step i, the sample is placed on a slide and coverslipped.
4. A kit for adipose tissue staining, the kit comprising:
a reagent bottle for containing or containing a phosphate buffer solution;
the reagent bottle is used for containing or containing TritonX-100;
the reagent bottle is used for containing or containing M buffer solution, wherein the M buffer solution comprises imidazole 50mmol/L, KCl 50mmol/L, MgCl2·6H2O 0.5mmol/L、EGTA 1.0mmol/L、EDTA·2H2O0.1 mmol/L, B-mercaptoethanol 1.0mmol/L, glycerol 4.0mmol/L, and M buffer solution pH 7.2;
a reagent bottle for containing or containing glutaraldehyde;
the reagent bottle is used for containing or containing oil red O;
a reagent bottle for containing or containing isopropanol;
a reagent bottle for containing or containing DMSO;
a reagent bottle for containing or containing Coomassie brilliant blue R250;
the reagent bottle is used for containing or containing distilled water;
a glass slide;
a cover glass;
the kit is used in the method of adipose tissue staining according to any one of claims 1 to 3.
5. A method of adipose tissue staining, the staining method comprising the steps of:
a. treating frozen sections of adipose tissue with DMSO;
b. removing the DMSO;
c. processing the frozen sections of the adipose tissues by using TritonX-100;
d. removing the TritonX-100;
e. fixing the frozen sections of adipose tissue with glutaraldehyde;
f. removing glutaraldehyde;
g. staining the frozen sections of adipose tissue with coomassie brilliant blue R250;
h. and (6) decoloring.
6. The method of adipose tissue staining of claim 5, wherein:
rinsing the frozen sections of adipose tissue with phosphate buffer prior to step a;
the thickness of the frozen section of the adipose tissue is 30-40 mu m;
in step a, treating the frozen section of the adipose tissue with 45-55% DMSO for 5-10 minutes;
in step c, processing frozen sections of the adipose tissues by 0.8-1.2% TritonX-100 for 15-25 min;
in step d, the TritonX-100 is removed with M buffer, wherein M buffer comprises imidazole 50mmol/L, KCl 50mmol/L, MgCl2·6H2O 0.5mmol/L、EGTA 1.0mmol/L、EDTA·2H2O0.1 mmol/L, B-mercaptoethanol 1.0mmol/L, glycerol 4.0mmol/L, and M buffer solution pH 7.2;
in step d, washing with M buffer solution for 1-3 times, each time for 3-6 min;
in step e, fixing the frozen sections of the adipose tissues with 2.5-3.5% glutaraldehyde for 20-40 min;
in step f, washing with phosphate buffer to remove glutaraldehyde;
in step g, staining the frozen sections of adipose tissue with 0.15-0.25% Coomassie Brilliant blue R250 for 25-35 min;
in step h, washing with distilled water for decolorization.
7. The method of adipose tissue staining of claim 6, wherein:
rinsing the frozen sections of adipose tissue three times for one minute each with phosphate buffer prior to step a;
the thickness of the frozen section of the adipose tissue is 35 mu m;
in step a, the frozen sections of adipose tissue are treated with 50% DMSO for 8 minutes;
in step c, the frozen sections of the adipose tissues are treated with 1% TritonX-100 for 20 min;
in step d, removing the TritonX-100 three times with M buffer, each time for 5 min;
in step e, the frozen sections of adipose tissue are fixed with 3% glutaraldehyde for 30 min;
in step f, washing three times with phosphate buffer to remove glutaraldehyde;
in step g, the frozen sections of adipose tissue were stained with 0.2% coomassie brilliant blue R250 for 30 min;
in the step h, washing with distilled water for two times for decolorization;
after step h, the sample was placed on a glass slide and coverslipped.
8. A kit for adipose tissue staining, the kit comprising:
a reagent bottle for containing or containing a phosphate buffer solution;
a reagent bottle for containing or containing DMSO;
the reagent bottle is used for containing or containing TritonX-100;
the reagent bottle is used for containing or containing M buffer solution, wherein the M buffer solution comprises imidazole 50mmol/L, KCl 50mmol/L, MgCl2·6H2O 0.5mmol/L、EGTA 1.0mmol/L、EDTA·2H2O0.1 mmol/L, B-mercaptoethanol 1.0mmol/L, glycerol 4.0mmol/L, and M buffer solution pH 7.2;
a reagent bottle for containing or containing glutaraldehyde;
a reagent bottle for containing or containing Coomassie brilliant blue R250;
the reagent bottle is used for containing or containing distilled water;
a glass slide;
a cover glass;
the kit is used in the method of adipose tissue staining according to any one of claims 5 to 7.
9. The method of any one of claims 1-3, or the use or use of the kit of claim 4, for simultaneously characterizing adipocyte lipid droplets in relation to a cytoskeleton or the adipocyte lipid droplets in relation to the cytoskeleton.
10. Use or use of the method of any one of claims 5-7, or the kit of claim 8, for characterizing the cytoskeleton of adipocytes.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910797148.5A CN110426264B (en) | 2019-08-27 | 2019-08-27 | Method for staining fat cells and application thereof |
| LU101831A LU101831B1 (en) | 2019-08-27 | 2020-06-04 | A Method for Staining Adipocytes and Application Thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910797148.5A CN110426264B (en) | 2019-08-27 | 2019-08-27 | Method for staining fat cells and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110426264A CN110426264A (en) | 2019-11-08 |
| CN110426264B true CN110426264B (en) | 2022-03-22 |
Family
ID=68417708
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910797148.5A Active CN110426264B (en) | 2019-08-27 | 2019-08-27 | Method for staining fat cells and application thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN110426264B (en) |
| LU (1) | LU101831B1 (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004028442A2 (en) * | 2002-09-26 | 2004-04-08 | Pfizer Products Inc. | Use of excipients to increase dna uptake by swine muscle cells |
| EP1690870B1 (en) * | 2003-11-05 | 2012-03-28 | Immuno-Biological Laboratories Co., Ltd. | Marker peptide for alzheimer's disease |
| CN102933705A (en) * | 2009-12-17 | 2013-02-13 | 金斯顿女王大学 | Decellularized adipose tissue |
| CN105738182A (en) * | 2016-02-25 | 2016-07-06 | 河南中医学院 | Fluorescent staining method for observing plant microstructure |
| CN110157805A (en) * | 2019-05-22 | 2019-08-23 | 北京大学 | A kind of HOXB13 joint target gene detection prediction adenocarcinoma of lung progress, prognosis and drug resistant kit downstream |
| WO2020041781A1 (en) * | 2018-08-23 | 2020-02-27 | Essenlix Corporation | Enhanced, rapid, homogeneous, cell staining and assay |
-
2019
- 2019-08-27 CN CN201910797148.5A patent/CN110426264B/en active Active
-
2020
- 2020-06-04 LU LU101831A patent/LU101831B1/en active IP Right Revival
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004028442A2 (en) * | 2002-09-26 | 2004-04-08 | Pfizer Products Inc. | Use of excipients to increase dna uptake by swine muscle cells |
| EP1690870B1 (en) * | 2003-11-05 | 2012-03-28 | Immuno-Biological Laboratories Co., Ltd. | Marker peptide for alzheimer's disease |
| CN102933705A (en) * | 2009-12-17 | 2013-02-13 | 金斯顿女王大学 | Decellularized adipose tissue |
| CN105738182A (en) * | 2016-02-25 | 2016-07-06 | 河南中医学院 | Fluorescent staining method for observing plant microstructure |
| WO2020041781A1 (en) * | 2018-08-23 | 2020-02-27 | Essenlix Corporation | Enhanced, rapid, homogeneous, cell staining and assay |
| CN110157805A (en) * | 2019-05-22 | 2019-08-23 | 北京大学 | A kind of HOXB13 joint target gene detection prediction adenocarcinoma of lung progress, prognosis and drug resistant kit downstream |
Non-Patent Citations (3)
| Title |
|---|
| "Human liver fatty aldehyde dehydrogenase: microsomal localization, purification, and biochemical characterization";Todd LKelson 等;《Biochimica et Biophysica Acta (BBA) - General Subjects》;19971231;第1335卷(第1-2期);第99-110页 * |
| "考马斯亮蓝染色法观察细胞微丝";无;《百度文库》;20190107;第1-13页 * |
| "胰岛素和肾上腺素对前体脂肪细胞增殖与分化的调控";孙超 等;《Animal Biotechnology Bulletin》;20041231;第9卷(第1期);第204-209页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| LU101831B1 (en) | 2021-04-29 |
| CN110426264A (en) | 2019-11-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104155160A (en) | Tissue dewaxing transparent agent free of benzene | |
| US11415494B2 (en) | Method of rapidly and completely rendering lipid-droplet-rich tissue transparent | |
| Pedersen | Studies on the nature of planarian connective tissue | |
| JPWO2014069519A1 (en) | Clarified biological specimen preparation kit and transparent biological specimen preparation method | |
| CN105738182A (en) | Fluorescent staining method for observing plant microstructure | |
| Kelly et al. | The metachromatic reaction | |
| Moore et al. | Low temperature sulfation of tissues and the demonstration of metachromasy | |
| Nordenstam et al. | Chromaffin granules and their cellular location in human skin | |
| CN110426264B (en) | Method for staining fat cells and application thereof | |
| Maegolena | Feulgen's reaction and some of its applications for botanical material | |
| CN110596096B (en) | Transparentizing reagent, application of transparentizing reagent in optical imaging of biological tissue material and living skin tissue transparentizing imaging method | |
| CN104872110A (en) | Crystal skeleton specimen manufacturing method | |
| CN101897328B (en) | Method for staining protozoon sample | |
| BR112012001495A2 (en) | composition of cytological or histological fixation and staining processes | |
| CN107058593A (en) | A kind of hypotype Ph likeALL detection method | |
| CN105504873A (en) | Dye composition for nervous tissue nissl body dyeing, dye liquor and dyeing method | |
| CN107063811B (en) | Dyeing method for observing collagen fibers by adopting polarized light microscope | |
| CN109900537A (en) | A kind of processing method of histological tissue specimen | |
| JPH03167472A (en) | Method for dyeing embryo skeletal of experimental animal | |
| CN105648035B (en) | Alpha-naphthol acetate esterase staining kit | |
| Viola et al. | Connective tissue stains-A review | |
| Yasui | Ethyl alcohol as a fixative for smear materials | |
| Yu et al. | Ultrafast aqueous clearing methods for 3D imaging | |
| GB2372811A (en) | Staining physiological samples | |
| Swartz et al. | Direct microscopic examination of the skin: a method for the determination of the presence of fungi |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20231018 Address after: 518 Yantian North Industrial Street, Shenzhen Patentee after: Shenzhen Huada gene cell technology Co.,Ltd. Address before: 236037 Qinghe West Road, Fuyang, Anhui Province, No. 100 Patentee before: Fuyang Normal University |