CN106912480A - A kind of kit preserved for tissue freezing section - Google Patents
A kind of kit preserved for tissue freezing section Download PDFInfo
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- CN106912480A CN106912480A CN201710190299.5A CN201710190299A CN106912480A CN 106912480 A CN106912480 A CN 106912480A CN 201710190299 A CN201710190299 A CN 201710190299A CN 106912480 A CN106912480 A CN 106912480A
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- Prior art keywords
- cup
- freezing section
- tissue freezing
- kit
- tissue
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0236—Mechanical aspects
- A01N1/0263—Non-refrigerated containers specially adapted for transporting or storing living parts whilst preserving, e.g. cool boxes, blood bags or "straws" for cryopreservation
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B65—CONVEYING; PACKING; STORING; HANDLING THIN OR FILAMENTARY MATERIAL
- B65D—CONTAINERS FOR STORAGE OR TRANSPORT OF ARTICLES OR MATERIALS, e.g. BAGS, BARRELS, BOTTLES, BOXES, CANS, CARTONS, CRATES, DRUMS, JARS, TANKS, HOPPERS, FORWARDING CONTAINERS; ACCESSORIES, CLOSURES, OR FITTINGS THEREFOR; PACKAGING ELEMENTS; PACKAGES
- B65D81/00—Containers, packaging elements, or packages, for contents presenting particular transport or storage problems, or adapted to be used for non-packaging purposes after removal of contents
- B65D81/18—Containers, packaging elements, or packages, for contents presenting particular transport or storage problems, or adapted to be used for non-packaging purposes after removal of contents providing specific environment for contents, e.g. temperature above or below ambient
- B65D81/22—Containers, packaging elements, or packages, for contents presenting particular transport or storage problems, or adapted to be used for non-packaging purposes after removal of contents providing specific environment for contents, e.g. temperature above or below ambient in moist conditions or immersed in liquids
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Environmental Sciences (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Mechanical Engineering (AREA)
- Physiology (AREA)
- Biophysics (AREA)
- Hematology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of kit preserved for tissue freezing section, including liquid and sample storage cup are preserved, the preservation liquid includes following components：Sodium chloride 75mM, potassium chloride 1.5mM, disodium hydrogen phosphate 5.6mM, potassium dihydrogen phosphate 1.0mM, sodium azide 3.1mM, ethylenediamine tetra-acetic acid 2mM, 45% glycerine.The kit preserved for tissue freezing section of the invention, prepares simple, and the preparation raw material for preserving liquid is conventional biochemical reagents, it is easy to use, only there need to be refrigerator, improve the utilization rate of precious sample, be that research work saves human cost and material cost.
Description
Technical field
The invention belongs to technical field of molecular biology, more particularly to a kind of reagent preserved for tissue freezing section
Box.
Background technology
Histotomy is most morphologic bases, either Clinicopathologic Diagnosis or basic medical research, all
It is unable to do without the support of tectology.Conventional histotomy mainly includes frozen section and paraffin section, both advantage and disadvantage
It is mainly reflected in the following aspects：1st, frozen section is simple and quick, can preferably preserve tissue antigen, but be also easy to produce ice
Crystalline substance destruction tissue cellularity, and histotomy can not be too thin (10-100 μm)；2nd, paraffin section can cut tissue very
Thin (3-5 μm)；But it is cumbersome time-consuming, often result in the loss of tissue antigen.
After tissue soaks through sucrose, the generation of ice crystal in being organized during frozen section can be avoided.Simultaneously because copolymerization is burnt aobvious
The invention of micro- imaging, tissue cellularity also can be clearly presented when even for thicker histotomy.Therefore in many
In life science work, especially neuroscience field, frozen section is increasingly becoming leading histotomy.
Though tissue freezing section there are above-mentioned many advantages, also there are following fatal defects in actual research work：
Tissue block (such as 1mm3Size) hundreds of sections can be usually cut out, but the histotomy of ten or so once need to be only used, remain
Under most sections have little time effectively utilization, be primarily due to that these histotomies are interior at one week to rot, even if
It is to add the preservatives such as Sodium azide also to be difficult to preserve one month.If unessential tissue, there is this shortcoming unimportant；
But for precious research material, the waste of any histotomy seems particularly unfortunate.
In view of this, it is necessary to which a kind of kit that can for a long time preserve tissue freezing section is provided.
The content of the invention
For defect of the prior art, it is an object of the invention to provide a kind of reagent preserved for tissue freezing section
Box.
To achieve these goals, the technical solution adopted by the present invention is as follows：
One aspect of the present invention provides a kind of kit preserved for tissue freezing section, including preserves liquid and storage
Sample cup.
The preservation liquid includes following components：Sodium chloride 75mM (mmol/L), potassium chloride 1.5mM, ten hydrogen phosphate dihydrates
Disodium 5.6mM, potassium dihydrogen phosphate 1.0mM, sodium azide 3.1mM, ethylenediamine tetra-acetic acid 2mM, 45% glycerine (Vol/
Vol)。
The sample storage cup includes the cup lid of cup and the supporting sealing of the cup, and the cup is opened with bottom, top
Mouth is more than the structure of bottom, and the cup lid is placed on when on the cup, there is the lid ear for extending cup on the cup lid.
The cup top is provided with external screw thread, is sealed with the internal thread that the cup lid is provided with.
The cup is cylindrical or square.
The cup lid is flexible glue top cover.
The material of the cup is clear hard plastics.
The capacity of the cup is 12-17mL.
When the cup is for cylinder, its base diameter is 1.3-1.8cm, is highly 2.0-2.8cm, and top diameter is
1.4-2.0cm, is highly 1.5-1.7 times of base diameter.
The amount for preserving liquid is 200mL.
The amount of the sample storage cup is 100.
There is advantages below and beneficial effect due to using above-mentioned technical proposal, the present invention：
The kit preserved for tissue freezing section of the invention, prepares simple, and the preparation raw material for preserving liquid is often
The biochemical reagents of rule, it is easy to use, only there need to be refrigerator, the utilization rate of precious sample is improve, it is that research work is saved
Human cost and material cost.
The kit preserved for tissue freezing section of the invention, make use of glycerine can fresh-keeping characteristic, by its with
Phosphate buffer is mixed by proper proportion, while adding other preservatives and metal ion chelation agent, makes it permanent
Ground preserves tissue freezing section's (being up to more than 2 years), and its relatively low viscosity is also convenient for researcher and uses.
Brief description of the drawings
Fig. 1 be the embodiment of the present invention the kit preserved for tissue freezing section in the structure of cup of sample storage cup show
It is intended to.
Fig. 2 be the embodiment of the present invention the kit preserved for tissue freezing section in the structure of cup lid of sample storage cup show
It is intended to.
Wherein：1 is cup, and 2 is cup lid, and 3 is lid ear.
Specific embodiment
In order to illustrate more clearly of the present invention, with reference to preferred embodiment, the present invention is described further.Ability
Field technique personnel should be appreciated that following specifically described content is illustrative and be not restrictive, and should not limit this with this
The protection domain of invention.
The embodiment of the present invention is raw materials used as follows：Sodium chloride (NaCl), purchased from Chemical Reagent Co., Ltd., Sinopharm Group；Chlorine
Change potassium (KCl), purchased from Chemical Reagent Co., Ltd., Sinopharm Group；Disodium hydrogen phosphate (Na2HPO4·12H2O), it is purchased from
Chemical Reagent Co., Ltd., Sinopharm Group；Potassium dihydrogen phosphate (KH2PO4), purchased from Chemical Reagent Co., Ltd., Sinopharm Group；The third three
Alcohol (C3H8O3), purchased from Sigma companies；Sodium azide (NaN3), purchased from Sigma companies；Ethylenediamine tetra-acetic acid (EDTA), is purchased from
Chemical Reagent Co., Ltd., Sinopharm Group.
Embodiment 1
A kind of kit preserved for tissue freezing section, including liquid 200mL is preserved, the bag (100) of sample storage cup one makes
It is a with specification.
The formula for preserving liquid is as follows：NaCl 75mM, KCl 1.5mM, Na2HPO4·12H2O 5.6mM, KH2PO41.0mM,
NaN33.1mM, EDTA 2mM, 45% glycerine (Vol/Vol)；It is dissolved in deionized water, filters, deaeration in condenser, 4 DEG C is kept away
Light is preserved.
The preparation method for preserving liquid is comprised the following steps：
The first step, by 8 grams of NaCl, 0.2 gram of KCl, 3.63 grams of Na2HPO4·12H2O, 0.24 gram of KH2PO4, 0.37 gram of NaN3
It is separately added into 800 milliliters of deionized water with 10.6 grams of EDTA, and being stirred with glass bar makes it fully dissolve, then constant volume is arrived
1000 milliliters.
Second step, glycerine of the above-mentioned buffer solution with 90 milliliters for taking 110 milliliters is fully mixed.
3rd step, with 0.4 micron of filter (millipore, the U.S.) filter, then with vavuum pump (Beijing 61, in
State) deoxygenation, then seal and be put into 4 DEG C of refrigerators and keep in dark place.
Topmost fresh-keeping composition is glycerine in preserving liquid, and its concentration has preservation when being more than 30%, with
Its fresh-keeping effect of the raising of concentration is stronger, but concentration is got over high viscosity increase and is unfavorable for using.Choosing comprehensively pros and cons, its is dense
Degree is set as 45%, using effect preferably (even if experiment proves that the tissue for preserving more than 3 years is not damaged yet).Herein molten
Agent is phosphate buffer, and other isotonic solutions (such as trishydroxymethylaminomethane-hydrochloride buffer, physiological saline) can also be examined
Consider, but in these all possible formulas, technical scheme relative efficacy is best.
Sample storage cup includes cup 1 and the cup in the kit preserved for tissue freezing section of the embodiment of the present invention
The cup lid 2 of 1 supporting sealing, the cup 1 is the structure for being more than bottom with bottom, open top, and the cup lid 2 is placed on described
When on cup 1, there is the lid ear 3 for extending cup 1 on the cup lid 2；As depicted in figs. 1 and 2, Fig. 1 is this to its structural representation
The structural representation of the cup of sample storage cup in the kit preserved for tissue freezing section of inventive embodiments, Fig. 2 is this hair
The structural representation of the cup lid of sample storage cup in the kit preserved for tissue freezing section of bright embodiment.
In the present embodiment, the top of cup 1 is provided with external screw thread, the internal thread sealing being provided with cup lid 2, the capacity of cup 1
It is 12-17mL, material is clear hard plastics, and the shape of cup 1 can be cylindrical or square, when it is cylinder, bottom
The a diameter of 1.3-1.8cm in portion, is highly 2.0-2.8cm, and top diameter is 1.4-2.0cm, is highly the 1.5-1.7 of base diameter
Times, the preferred base diameter of sample storage cup is 1.5cm, and top diameter is 1.8cm, is highly 2.5cm, and cup lid 2 is flexible glue top cover.
In the present embodiment, the capacity of cup 1 is 12-17mL, and sample storage cup is flat, transparent, rigid plastics, wide-mouth sets
Meter.Existing product is not dedicated for the small size container of histotomy, though other small size reagent bottles can be temporarily borrowed, big rule
Mould is not very convenient using still.Sample storage cup in the present embodiment is to aim at tissue to preserve for a long time and design, and is not only preserved
Histotomy is taken conveniently, and is also very suitable for the high-volume in laboratory and is used.
The application method of the kit preserved for tissue freezing section is as follows：For the tissue that needs carry out long-term preservation
Frozen section, is transferred to sample storage cup, and three times (3 × 5 minutes) are washed with PBS by the section for needing to preserve first；Then
Blot PBS and add 4mL to preserve liquid and mix；Last closed, 4 DEG C keep in dark place.
Embodiment 2
As a example by the preservation of 15 monthly age Alzheimer disease (AD) transgenic mouse brain tissues section and taking.
The first step, tissue freezing section is as follows：
1st, freezing microtome temperature (- 18 DEG C of specimen holder fixator, -20 DEG C of cabinet), 30 microns of slice thickness are set；
2nd, Mice brain tissues are carried out into coronal-plane serial section, and is divided into 6 equal portions.
Second step, the washing and preservation of brain tissue slice is as follows：
1st, 6 parts of sections are transferred in 6 sample storage cups respectively, 3-4 milliliters of phosphate buffer is added in each sample storage cup
(PBS) record of brain piece information, and in cup body is carried out；
2nd, PBS is washed three times, every time five minutes；
3rd, the PBS in cup is blotted, 2-3 milliliters of preservation liquid is subsequently adding and is rocked mixing with histotomy；
4th, cup lid is covered, the refrigerator that temperature is 4 DEG C is then placed in and is kept in dark place.
3rd step, preserves taking for brain tissue as follows：
1st, the histotomy (depending on party's concrete condition) is opened with picking 4-6 in eyebrow pencil therefrom a sample storage cup to put
Enter to fill in the glass dish (Puri beautiful this, Wuhan) of PBS, then made a record on sample storage cup and put back to refrigerator and continue to protect
Deposit；
2nd, the histotomy of taking-up is washed three times, every time five minutes with PBS；
3rd, washed histotomy can be normally carried out follow-up dying operation.
Embodiment 3
So that the brain tissue slice of mesencephalic arteries embolism mouse ischemia model is preserved as an example.
The first step, tissue freezing section is as follows：
1st, freezing microtome temperature (- 18 DEG C of specimen holder fixator, -20 DEG C of cabinet), 30 microns of slice thickness are set；
2nd, Mice brain tissues are carried out into coronal-plane serial section, and is divided into 6 equal portions.
Second step, the washing and preservation of brain tissue slice is as follows：
1st, 6 parts of sections are transferred in 6 sample storage cups respectively, 3-4 milliliters of phosphate buffer is added in each sample storage cup
(PBS) record of brain piece information, and in cup body is carried out；
2nd, PBS is washed three times, every time five minutes；
3rd, the PBS in cup is blotted, 2-3 milliliters of preservation liquid is subsequently adding and is rocked mixing with histotomy；
4th, cup lid is covered, the refrigerator that temperature is 4 DEG C is then placed in and is kept in dark place.
3rd step, preserves taking for brain tissue as follows：
1st, the histotomy (depending on party's concrete condition) is opened with picking 4-6 in eyebrow pencil therefrom a sample storage cup to put
Enter to fill in the glass dish (Puri beautiful this, Wuhan) of PBS, then made a record on sample storage cup and put back to refrigerator and continue to protect
Deposit；
2nd, the histotomy of taking-up is washed three times, every time five minutes with PBS；
3rd, washed histotomy can be normally carried out follow-up dying operation.
Embodiment 4
So that the frozen section of human glioma is preserved as an example.
The first step, the fixation of glioma pathological tissue and sucrose immersion are as follows：
1st, it is fixed：The pathological tissue that takes out of performing the operation is dipped into 4% paraformaldehyde solution (Puri beautiful this, Wuhan) immediately
In, and be put into the refrigerator that temperature is 4 DEG C and preserve 48 hours；
2nd, sink to the bottom：Tissue after fixation is transferred to fill 30% sucrose (Puri it is beautiful this, Wuhan；About 8-10 milliliters) sample storage
In cup, and it is put into the refrigerator that temperature is 4 DEG C until tissue sinks to the bottom (about 48 hours).
Second step, tissue freezing section is as follows：
1st, freezing microtome temperature (- 18 DEG C of specimen holder fixator, -20 DEG C of cabinet), 30 microns of slice thickness are set；
2nd, Mice brain tissues are carried out into coronal-plane serial section, and is divided into 6 equal portions.
3rd step, the washing and preservation of brain tissue slice is as follows：
1st, 6 parts of sections are transferred in 6 sample storage cups respectively, 3-4 milliliters of phosphate buffer is added in each sample storage cup
(PBS) record of brain piece information, and in cup body is carried out；
2nd, PBS is washed three times, every time five minutes；
3rd, the PBS in cup is blotted, 2-3 milliliters of preservation liquid is subsequently adding and is rocked mixing with histotomy；
4th, cup lid is covered, the refrigerator that temperature is 4 DEG C is then placed in and is kept in dark place.
4th step, preserves taking for brain tissue as follows：
1st, the histotomy (depending on party's concrete condition) is opened with picking 4-6 in eyebrow pencil therefrom a sample storage cup to put
Enter to fill in the glass dish (Puri beautiful this, Wuhan) of PBS, then made a record on sample storage cup and put back to refrigerator and continue to protect
Deposit；
2nd, the histotomy of taking-up is washed three times, every time five minutes with PBS；
3rd, washed histotomy can be normally carried out follow-up dying operation.
Obviously, the above embodiment of the present invention is only intended to clearly illustrate example of the present invention, and is not right
The restriction of embodiments of the present invention, for those of ordinary skill in the field, may be used also on the basis of the above description
To make other changes in different forms, all of implementation method cannot be exhaustive here, it is every to belong to this hair
Obvious change that bright technical scheme is extended out changes row still in protection scope of the present invention.
Claims (10)
1. it is a kind of for tissue freezing section preserve kit, it is characterised in that：Including preserving liquid and sample storage cup.
2. it is according to claim 1 for tissue freezing section preserve kit, it is characterised in that：The preservation liquid bag
Include following components：It is sodium chloride 75mM, potassium chloride 1.5mM, disodium hydrogen phosphate 5.6mM, potassium dihydrogen phosphate 1.0mM, folded
Sodium nitride 3.1mM, ethylenediamine tetra-acetic acid 2mM, 45% glycerine.
3. it is according to claim 1 for tissue freezing section preserve kit, it is characterised in that：The sample storage cup bag
The cup lid of cup and the supporting sealing of the cup is included, the cup is the structure for being more than bottom with bottom, open top, described
Cup lid is placed on when on the cup, there is the lid ear for extending cup on the cup lid.
4. it is according to claim 3 for tissue freezing section preserve kit, it is characterised in that：The cup top
External screw thread is provided with, is sealed with the internal thread that the cup lid is provided with.
5. it is according to claim 3 for tissue freezing section preserve kit, it is characterised in that：The cup is circle
Cylindricality is square.
6. it is according to claim 3 for tissue freezing section preserve kit, it is characterised in that：The cup lid is soft
Glue top cover.
7. it is according to claim 3 for tissue freezing section preserve kit, it is characterised in that：The material of the cup
Matter is clear hard plastics.
8. it is according to claim 3 for tissue freezing section preserve kit, it is characterised in that：The appearance of the cup
It is 12-17mL to measure.
9. it is according to claim 5 for tissue freezing section preserve kit, it is characterised in that：The cup is circle
During cylindricality, its base diameter is 1.3-1.8cm, is highly 2.0-2.8cm, and top diameter is 1.4-2.0cm, highly for bottom is straight
1.5-1.7 times of footpath.
10. it is according to claim 1 for tissue freezing section preserve kit, it is characterised in that：The preservation liquid
Amount be 200mL；
The amount of the sample storage cup is 100.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710190299.5A CN106912480A (en) | 2017-03-28 | 2017-03-28 | A kind of kit preserved for tissue freezing section |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710190299.5A CN106912480A (en) | 2017-03-28 | 2017-03-28 | A kind of kit preserved for tissue freezing section |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106912480A true CN106912480A (en) | 2017-07-04 |
Family
ID=59460463
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710190299.5A Pending CN106912480A (en) | 2017-03-28 | 2017-03-28 | A kind of kit preserved for tissue freezing section |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106912480A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5612218A (en) * | 1991-12-03 | 1997-03-18 | Busch; Christer | Method of handling biologic matter and means for carrying out the method |
-
2017
- 2017-03-28 CN CN201710190299.5A patent/CN106912480A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5612218A (en) * | 1991-12-03 | 1997-03-18 | Busch; Christer | Method of handling biologic matter and means for carrying out the method |
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Inventor after: Yan Huanhuan Inventor before: Yan Huanhuan Inventor before: Lu Youming |
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Application publication date: 20170704 |
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