EP1350101A2 - Preparation tissulaire ou cellulaire morphometrique - Google Patents
Preparation tissulaire ou cellulaire morphometriqueInfo
- Publication number
- EP1350101A2 EP1350101A2 EP01985910A EP01985910A EP1350101A2 EP 1350101 A2 EP1350101 A2 EP 1350101A2 EP 01985910 A EP01985910 A EP 01985910A EP 01985910 A EP01985910 A EP 01985910A EP 1350101 A2 EP1350101 A2 EP 1350101A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- weight
- cells
- tissue
- morphometric
- cell preparation
- 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.)
- Withdrawn
Links
- 238000002360 preparation method Methods 0.000 title claims abstract description 32
- 230000003562 morphometric effect Effects 0.000 title claims abstract description 25
- 238000013425 morphometry Methods 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 23
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 claims abstract description 16
- 239000011159 matrix material Substances 0.000 claims abstract description 15
- 239000012192 staining solution Substances 0.000 claims abstract description 15
- 210000004027 cell Anatomy 0.000 claims description 56
- 210000001519 tissue Anatomy 0.000 claims description 46
- 239000013543 active substance Substances 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 238000011156 evaluation Methods 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 7
- 239000003960 organic solvent Substances 0.000 claims description 7
- 238000010186 staining Methods 0.000 claims description 7
- 239000003086 colorant Substances 0.000 claims description 6
- 235000002639 sodium chloride Nutrition 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- -1 alicyclic alcohols Chemical class 0.000 claims description 5
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 4
- 238000011534 incubation Methods 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 239000002504 physiological saline solution Substances 0.000 claims description 4
- 210000004881 tumor cell Anatomy 0.000 claims description 4
- 230000033077 cellular process Effects 0.000 claims description 3
- 239000013078 crystal Substances 0.000 claims description 3
- 210000002241 neurite Anatomy 0.000 claims description 3
- 210000002569 neuron Anatomy 0.000 claims description 3
- HKJKONMZMPUGHJ-UHFFFAOYSA-N 4-amino-5-hydroxy-3-[(4-nitrophenyl)diazenyl]-6-phenyldiazenylnaphthalene-2,7-disulfonic acid Chemical compound OS(=O)(=O)C1=CC2=CC(S(O)(=O)=O)=C(N=NC=3C=CC=CC=3)C(O)=C2C(N)=C1N=NC1=CC=C([N+]([O-])=O)C=C1 HKJKONMZMPUGHJ-UHFFFAOYSA-N 0.000 claims description 2
- 102000008186 Collagen Human genes 0.000 claims description 2
- 108010035532 Collagen Proteins 0.000 claims description 2
- 102000016359 Fibronectins Human genes 0.000 claims description 2
- 108010067306 Fibronectins Proteins 0.000 claims description 2
- 102000007547 Laminin Human genes 0.000 claims description 2
- 108010085895 Laminin Proteins 0.000 claims description 2
- ZHAFUINZIZIXFC-UHFFFAOYSA-N [9-(dimethylamino)-10-methylbenzo[a]phenoxazin-5-ylidene]azanium;chloride Chemical compound [Cl-].O1C2=CC(=[NH2+])C3=CC=CC=C3C2=NC2=C1C=C(N(C)C)C(C)=C2 ZHAFUINZIZIXFC-UHFFFAOYSA-N 0.000 claims description 2
- 239000004480 active ingredient Substances 0.000 claims description 2
- 125000003118 aryl group Chemical group 0.000 claims description 2
- 210000000601 blood cell Anatomy 0.000 claims description 2
- 229920001436 collagen Polymers 0.000 claims description 2
- 210000002744 extracellular matrix Anatomy 0.000 claims description 2
- 238000007491 morphometric analysis Methods 0.000 claims description 2
- 210000000663 muscle cell Anatomy 0.000 claims description 2
- 210000004498 neuroglial cell Anatomy 0.000 claims description 2
- 210000003594 spinal ganglia Anatomy 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 2
- 210000004748 cultured cell Anatomy 0.000 claims 2
- 125000002723 alicyclic group Chemical group 0.000 claims 1
- 239000000427 antigen Substances 0.000 abstract description 4
- 102000036639 antigens Human genes 0.000 abstract description 4
- 108091007433 antigens Proteins 0.000 abstract description 4
- 239000000975 dye Substances 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 6
- 108010025020 Nerve Growth Factor Proteins 0.000 description 4
- 102000015336 Nerve Growth Factor Human genes 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 210000003850 cellular structure Anatomy 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 229940053128 nerve growth factor Drugs 0.000 description 3
- 208000003098 Ganglion Cysts Diseases 0.000 description 2
- 208000005400 Synovial Cyst Diseases 0.000 description 2
- 230000019552 anatomical structure morphogenesis Effects 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 230000001002 morphogenetic effect Effects 0.000 description 2
- 230000014511 neuron projection development Effects 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 238000003909 pattern recognition Methods 0.000 description 2
- 238000007447 staining method Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 241001417092 Macrouridae Species 0.000 description 1
- 238000006683 Mannich reaction Methods 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102000007072 Nerve Growth Factors Human genes 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 235000009233 Stachytarpheta cayennensis Nutrition 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000000981 basic dye Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000000512 collagen gel Substances 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 238000002135 phase contrast microscopy Methods 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 238000007493 shaping process Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
Classifications
-
- 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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5082—Supracellular entities, e.g. tissue, organisms
- G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
-
- 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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
Definitions
- the invention relates to a morphometric tissue or cell preparation which can be obtained by carrying out the following process steps: a) cells or tissue are cultivated in a matrix for a defined time under defined conditions, b) after the defined time has elapsed, the cells or cells are cultured Tissue-containing matrix incubated with a staining solution.
- the invention further relates to a manufacturing method for such a preparation and uses thereof.
- the invention relates to a fixing and staining solution for use in such a method.
- a morphometric tissue or cell preparation is treated in a manner that allows recognition of the
- Form formation of cellular structures or of tissue structures allowed. It is essential here that shapes, for example shapes of cells and / or cell components and / or tissues, are made recognizable by means of contrast-forming methods. Detection can be carried out by observation using various optical methods, in the simplest case using normal light microscopy. Often, certain shape features are counted as part of the recognition, which are usually correlated with certain cellular processes. The detection can be carried out visually by the human eye, with a person viewing then, for example, identifying sought structures and possibly counting them using the
- Pattern recognition system which can then automatically count.
- a recording can be carried out photographically or electronically, for example using CCD elements.
- the methodology is used to determine what influence the defined cultivation conditions have on morphogenesis.
- the expression of the defined cultivation conditions therefore also includes, in particular, non-natural or non-standard conditions, which are characterized, for example, by adding one or more prospective active ingredients to a standard medium. It is understood that the
- the coloring solution serves to highlight the shaping features of interest in contrast.
- the average person skilled in the art can easily select the dye according to the forms of interest (for example, basic dyes bind to acidic cell structures, such as DNA in the nucleus or RNA in the ribosomes; acidic dyes preferably bind to basic ones
- Dye then form the colored areas, while areas in which there are no or fewer binding sites for the dye are not or only weakly colored areas. Ultimately, this is the mechanism of contrast formation.
- a method of the type mentioned at the outset is, for example, from the literature reference Leclere, P., Neuroscience: 82, 545-548, 1998.
- ganglion cell explants were treated with a nerve growth factor or cultured in the presence thereof, and then this was induced
- Neurite growth analyzed morphometrically. Staining was carried out using dye-labeled or dye-labeled antibodies. It is therefore an antigen-dependent staining method. specific
- Stains of this type are only suitable for very thin matrices. However, it is often desirable to use distinctly three-dimensional matrices or cultures, in which case a sufficiently homogeneous antigen-dependent staining is difficult or impossible. In addition, antigen-dependent staining methods are time-consuming and expensive.
- the invention teaches a morphometric tissue or
- Cell preparation which can be obtained by carrying out the following process steps: a) cells or tissues are cultivated for a defined time under defined conditions in a three-dimensional matrix, b) after the defined time has elapsed, the matrix containing the cultivated cells or tissues is treated with a Fixing and staining solution containing an aldehyde and, optionally an alcohol, and an antigen-unspecific stain incubated. This can be followed by a washing process step, for example with water.
- An antigen-nonspecific colorant is a dye that can bind non-specifically to molecules or molecular classes of a cell or a tissue solely because of its acidic or basic groups, but also through redox processes. Binding to virtually all structures of cells or tissue can also take place.
- Antibodies are not used.
- the aldehyde and possibly the alcohol brings about a reliable fixation of structures of the cells or of the tissue by a variety of immobilizing reactions with intrinsically mobile molecules of the cells or of the tissue, for example by Mannich reactions.
- this includes preparations whose spatial extent in all three spatial directions is a multiple of the spatial extent of the cultivated cells, for example equal to or more than 2 times, preferably more than 10 times, up to more than 100 times , It is achieved with the invention that this is pronounced three-dimensional line or tissue preparations are stable and permanently fixed and at the same time very high-contrast images are obtained using the simplest methods, for example simple light microscopy. Only one treatment with the fixing and staining solution is necessary, ie it is a "one-step” method. In addition, pronounced three-dimensional specimens can be fixed and stained easily, while specific staining of such specimens using antibody-based methods is hardly possible, if at all. Pronounced three-dimensional preparations are those that do not only have a minimal thickness (cuts), but rather can have thicknesses from 0.2 ⁇ m to 20 mm, in particular 0.2 mm to 20 mm.
- the fixing and staining solution contains water, optionally a water-miscible organic solvent, and
- the aqueous saline solution reliably prevents changes in the shape of cells or tissue, for example due to osmosis, in the course of fixation and staining.
- the fixing and staining solution can contain the following components: A) 0.5-35% by weight, preferably 1-2% by weight, aldehyde, B) 0.01 to 10% by weight, preferably 0 , 3 - 0.6% by weight, colorant, C) 0 - 90% by weight, preferably 30 - 50% by weight, of one or more water-miscible organic solvents,
- E 0-5% by weight, preferably 0.1-1% by weight, of common salt, the sum of the proportions of components A) to E) always giving 100% by weight, and the ratio of the proportions by weight of Components E: D are in the range from 1: 1000 to 1: 100, preferably corresponds to the ratio by weight of a physiological saline solution.
- the invention also relates independently to such a fixing and staining solution.
- the duration of the incubation in stage b) can be in the range from 0.5 to 24 h, preferably in the range from 1 to 10 min.
- the incubation in stage b) can be carried out at a temperature from 0 ° C. to 60 ° C., preferably from 0 ° C. to 40 ° C.
- the water-miscible organic solvent can be selected from the group consisting of "Cl-10 alkanols with 1-3 OH groups, Cl-10 alicyclic alcohols with 1-3 OH groups, Cl to C10 aromatic alcohols, and mixtures of such substances ". Ethanol is preferred.
- the colorant can be selected from the group consisting of "crystal violet, amido black, cresyl violet, and mixtures of such substances”.
- the matrix can be selected from the group consisting of "extracted natural extracellular matrices of various origins, in particular from rat tails or pig skin, their main components, in particular collagen, laminin, fibronectin, and mixtures of these substances".
- the aldehyde can be selected from the group consisting of "Cl to C10 alkanaldehydes, Cl to C10 alicyclic aldehydes, aromatic Cl to C10 aldehydes, and mixtures of such aldehydes". Formaldehyde and paraformaldehyde are preferred.
- the individual cells or complex tissues of the preparation can be or consist of, for example, tumor cells, nerve cells, glial cells, muscle cells, blood cells. In the area of drug binding, the cells are optimally (but not necessarily) human cells.
- the invention further teaches a method of manufacture of a morphometric tissue or cell preparation, the following process steps being carried out: a) cells or tissues are cultivated over a defined time under defined conditions in a pronounced three-dimensional matrix, b) after the defined time has elapsed, the matrix containing the cultivated cells or tissue is included a fixing and staining solution containing an aldehyde and an antigen-unspecific stain.
- a) cells or tissues are cultivated over a defined time under defined conditions in a pronounced three-dimensional matrix
- the matrix containing the cultivated cells or tissue is included a fixing and staining solution containing an aldehyde and an antigen-unspecific stain.
- the invention furthermore teaches the use of a morphometric tissue or cell preparation according to the invention for the morphometric analysis of cellular processes caused by cultivation conditions.
- defined conditions are set from which a certain morphogenetic effect is expected. This is then evaluated and correlated with the defined conditions, if necessary semi-quantitative or quantitative.
- This also includes the use of a morphometric tissue or cell preparation according to the invention in a process for finding active substances, the active substance being used in step a), with the cells or tissue being subsequently recorded in step b) by means of, for example, an optical microscope, wherein the image obtained is preferably evaluated by means of an image recognition system, the result of the evaluation being assigned to the active substance, and where appropriate the method being repeated for different active substances and the results of the evaluation obtained for different active substances being compared with one another.
- a quantitative, or at least semi-quantitative, statement is obtained regarding the orphogenetic effect caused by the investigated active substances.
- the cells can, for example, ganglion cells, especially dorsal Schuwurzelganglien, be, the evaluation then z. B. includes the detection of newly formed neurites.
- the cells can also be tumor cells, the evaluation then comprising, for example, the detection of emigrated cells.
- the cultivation was then carried out for several hours in a culture medium containing nerve growth factor.
- the cultivation was ended by aspirating the culture medium from the gel and by the following staining and fixing step.
- Example 1 The gel obtained in Example 1 was then treated with a filtered fixing and staining solution. This contained: 2.42 g crystal violet, 162 ml ethanol (100%), 323 ml distilled water, 16 ml formaldehyde (35%, balance water), and 0.8 g NaCl. The treatment was carried out for 5 min. at 20 ° C. Then it was washed with water.
- the washed gel was then placed on a light microscope (Zeiss Axiovert, bright field, magnification of the objective lOx) and a detailed picture was taken, as shown in FIG. 1.
- a light microscope Zeiss Axiovert, bright field, magnification of the objective lOx
- FIG. 1 In the figure 1 below is the edge of the
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Toxicology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
La présente invention concerne une préparation tissulaire ou cellulaire morphométrique qui peut être obtenue grâce à la mise en oeuvre des étapes suivantes: a) des cellules ou des tissus sont cultivés pendant un temps défini dans des conditions définies dans une matrice tridimensionnelle; b) après écoulement du temps défini, la matrice contenant les cellules ou tissus cultivés est incubée avec une composition de fixation et de coloration contenant un aldéhyde ainsi qu'un colorant non antigène-spécifique.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE10101319 | 2001-01-09 | ||
| DE10101319A DE10101319A1 (de) | 2001-01-09 | 2001-01-09 | Morphometrisches Gewebe- oder Zellpräparat |
| PCT/EP2001/015055 WO2002055680A2 (fr) | 2001-01-09 | 2001-12-20 | Preparation tissulaire ou cellulaire morphometrique |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP1350101A2 true EP1350101A2 (fr) | 2003-10-08 |
Family
ID=7670432
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP01985910A Withdrawn EP1350101A2 (fr) | 2001-01-09 | 2001-12-20 | Preparation tissulaire ou cellulaire morphometrique |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US20040072281A1 (fr) |
| EP (1) | EP1350101A2 (fr) |
| JP (1) | JP2004520034A (fr) |
| AU (1) | AU2002235799A1 (fr) |
| DE (1) | DE10101319A1 (fr) |
| WO (1) | WO2002055680A2 (fr) |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5069374A (fr) * | 1973-10-24 | 1975-06-10 | ||
| DE3635711A1 (de) * | 1986-10-21 | 1988-04-28 | Knoll Ag | 5-nitrobenzo(de)isochinolin-1,3-dione, ihre herstellung und verwendung |
| DE3843534A1 (de) * | 1988-12-23 | 1990-07-12 | Basf Ag | Neue tnf-polypeptide |
| US5410016A (en) * | 1990-10-15 | 1995-04-25 | Board Of Regents, The University Of Texas System | Photopolymerizable biodegradable hydrogels as tissue contacting materials and controlled-release carriers |
| WO1998006390A1 (fr) * | 1996-08-13 | 1998-02-19 | Marigen S.A. | Concentres spontanement dispersibles d'esters de sterols et de derives de vitamine d a effet antiviral, virucide et/ou parasiticide |
-
2001
- 2001-01-09 DE DE10101319A patent/DE10101319A1/de not_active Withdrawn
- 2001-12-20 JP JP2002556729A patent/JP2004520034A/ja active Pending
- 2001-12-20 EP EP01985910A patent/EP1350101A2/fr not_active Withdrawn
- 2001-12-20 WO PCT/EP2001/015055 patent/WO2002055680A2/fr not_active Ceased
- 2001-12-20 AU AU2002235799A patent/AU2002235799A1/en not_active Abandoned
- 2001-12-20 US US10/250,782 patent/US20040072281A1/en not_active Abandoned
Non-Patent Citations (1)
| Title |
|---|
| See references of WO02055680A2 * |
Also Published As
| Publication number | Publication date |
|---|---|
| US20040072281A1 (en) | 2004-04-15 |
| DE10101319A1 (de) | 2002-07-18 |
| WO2002055680A3 (fr) | 2002-10-31 |
| WO2002055680A2 (fr) | 2002-07-18 |
| AU2002235799A1 (en) | 2002-07-24 |
| JP2004520034A (ja) | 2004-07-08 |
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