CN110286019A - A kind of whole embryo fixing means of striping anisolecithal egg - Google Patents
A kind of whole embryo fixing means of striping anisolecithal egg Download PDFInfo
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- CN110286019A CN110286019A CN201910679581.9A CN201910679581A CN110286019A CN 110286019 A CN110286019 A CN 110286019A CN 201910679581 A CN201910679581 A CN 201910679581A CN 110286019 A CN110286019 A CN 110286019A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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Abstract
The present invention provides a kind of whole embryo fixing means of striping anisolecithal egg, is that anisolecithal egg is placed in progress room temperature in trichloroacetic acid solution to fix；Remove trichloroacetic acid solution, anisolecithal egg is fixed with paraformaldehyde solution again；With phosphate buffer solution (PBS) replacing section paraformaldehyde fixer, 4 DEG C are saved for use；Pole likes that the fertilization membrane of the fertilized eggs after your fixation removes again, obtains complete embryo, is saved with phosphate buffer or methanol, is used for subsequent whole embryo immunohistochemistry or cytology correlative study.Anisolecithal egg after fixing means of the invention is fixed, embryo's flexible, when striping, are non-breakable, are easy to get complete embryo.
Description
Technical field
The invention belongs to the fixed studying technological domains of embryo, and in particular to a kind of whole embryo fixing means of striping anisolecithal egg.
Background technique
Immunohistochemistry technique or immunocytochemical technique are to study specific protein using Ag-Ab specific reaction
Qualitative, positioning and relative quantification expression effective ways of the matter in histocyte.
Sample fixation is the primary key link of immunohistochemical analysis.From the angle of immunohistochemistry technique, fixed effect
It is to solidify intracellular protein, reduces or terminate the reaction of exogenous enzymes and endogenous enzyme to the greatest extent；The self-dissolving of cell is prevented, with
Exempt from that antigen is made to diffuse to tissue interstitial；Keep the intrinsic form and structure of tissue；It is prior to be to maintain the anti-of tissue or cell
Originality inactivate antigen will not, diffusing phenomenon also do not occur, and reduce the background colour of immunohistochemical staining.
The fixed main purpose of sample is to save the intrinsic substance of cell, consolidating in solidification or sedimentation cell or in tissue fluid
Have substance, as nucleus, endoplasmic reticulum, mitochondria, Golgi's organs, lysosome, cross oxysome, cytoskeleton, tissue fluid, antigen etc.,
State of matter when cell or tissue being made to be kept substantially life.
But anisolecithal egg is in polarity distribution, and yolk content is very high, concentrates on plant pole；Wherein plasm is less, and main collection
In in animal pole.Immunohistochemical analysis is carried out to anisolecithal egg, such as using conventional fixing means such as 4% paraformaldehyde fixation side
Blastodisc after fixation can be completely stripped out by method although preferable for blastodisc part fixed effect, carry out immunohistochemistry point
Analysis, but 4% paraformaldehyde cannot be such that the Yolk of liquid solidifies, and cannot get complete embryo after fixed, be not easy to carry out whole
Embryo immunohistochemical analysis.Acetone and methanol have solidification to liquid yolk, for removing the solidification effect of the fertilized eggs after fertilization membrane
Fruit is preferable, available complete embryo.But for not going the fertilized eggs solidification effect of fertilization membrane poor, if using acetone and
Striping again after methanol is fixed, just cannot get complete embryo.
It is combined to enter inside egg cell convenient for high molecular weight protein antibody with testing protein (antigen), immunohistochemistry technique exists
Application in terms of egg cell is both needed to remove fertilization membrane.State of matter when in order to make cell or tissue be kept substantially life,
It is both needed to instant fixation.Therefore, fix, be not easily achieved again after the fertilization membrane of ovum living being removed, particularly with fertilization membrane with
The narrow anisolecithal egg of perivitelline between vitellinae membrana, it is necessary to remove fertilization membrane again after fixed.It is yellow in view of conventional fastening method fixing end
Ovum is unable to get the complete embryo of striping, needs to establish a kind of whole embryo fixing means of striping anisolecithal egg, is convenient for whole embryo and exempts from
Epidemic disease group or cytology correlative study.
Summary of the invention
For conventional fastening method for anisolecithal egg it is fixed present in deficiency, the present invention provides a kind of striping anisolecithal egg
Whole embryo fixing means is able to solve the whole embryo immunohistochemistry or cytology correlative study needs of anisolecithal egg.
The whole embryo fixing means of striping anisolecithal egg provided by the present invention, comprising the following steps:
1) trichloroacetic acid is fixed: anisolecithal egg being placed in progress room temperature in trichloroacetic acid solution and is fixed；
The concentration of the trichloroacetic acid solution is 4-6%；
The room temperature set time is fixed 0.5-2h；
2) paraformaldehyde is fixed: removing trichloroacetic acid solution, anisolecithal egg is fixed with paraformaldehyde solution again；
The concentration of the paraformaldehyde solution is 4%；
3) it replaces fixer: using phosphate buffer solution (PBS) replacing section paraformaldehyde fixer, 4 DEG C save for use；
The component of the phosphate buffer solution (PBS): 128mM NaCl, 2mM KCl, 8mM NaH2PO4,2mM
KH2PO4,pH 7.2。
4) striping: the fertilization membrane of the fertilized eggs after will be fixed in 3) removes, and obtains complete embryo, with phosphate buffer or
Methanol saves, and is used for subsequent whole embryo immunohistochemistry or cytology correlative study.
Further, above-mentioned fixing means, specifically includes the following steps:
1) trichloroacetic acid is fixed:
Anisolecithal egg is sucked in centrifuge tube, the solution of trichloroacetic acid of addition 4-6% after solution, the fixed 0.5-2 of room temperature are removed
Hour；
Preferably, being to be fixed using the trichloroacetic acid of 6% concentration 0.5 hour or the trichloroacetic acid of 4% concentration fixes 1
Hour；
2) paraformaldehyde is fixed: 4% paraformaldehyde solution, 4 DEG C of fixations are added in removal solution of trichloroacetic acid in anisolecithal egg
Overnight or room temperature is 2-3 hours fixed；
3) it replaces fixer: after paraformaldehyde has been fixed, replacing corresponding 3/4-4/5 volume with phosphate buffer solution (PBS)
Paraformaldehyde solution, and save in 4 DEG C stand-by；
4) striping: the fertilization membrane of step 3) treated fertilized eggs is removed, and obtains fixed complete embryo.
Compared with the method for the fixed fertilized eggs of common 4% paraformaldehyde, advantages of the present invention is as follows:
1. fixing means of the invention can be such that the Yolk in anisolecithal egg solidifies, available complete embryo after striping
Tire is particularly suitable for the fertilization higher anisolecithal egg of yolk content early period.
The anisolecithal egg after 2. fixing means of the invention is fixed, the perivitelline between fertilization membrane and vitellinae membrana increases, and is convenient for
Remove fertilization membrane by hand, is particularly suitable for the less anisolecithal egg of perivitelline.
The anisolecithal egg after 3. fixing means of the invention is fixed, embryo's flexible, when striping, is non-breakable, has been easy to get
Whole embryo.
4. this method has wide range of applications, 2- cell, 4- cell, 8- cell are arrived before being formed using this method to blastodisc
The equal higher anisolecithal egg of yolk contents is fixed, and available complete embryo after striping is convenient for whole embryo immunohistochemistry
Or cytology correlative study.
Detailed description of the invention
Fig. 1: 4% trichloroacetic acid fixes 2 hours, the overnight figure of 4% 4 DEG C of paraformaldehyde fixation, and wherein a is embryo after fixing
Tire, b are the embryo after striping；
Fig. 2: 6% trichloroacetic acid fixes 0.5 hour, and 4% 4 DEG C of paraformaldehyde fixation is schemed overnight, and wherein a is embryo after fixing
Tire, b are the embryo after striping；
Fig. 3: 6% trichloroacetic acid fixes 1 hour, and 4% 4 DEG C of paraformaldehyde fixation is overnight.A is embryo after fixing, and b is striping
Embryo afterwards.
Specific embodiment
Fixing means of the invention, specifically includes the following steps:
1) trichloroacetic acid is fixed:
Anisolecithal egg is sucked in centrifuge tube, the solution of trichloroacetic acid of addition 4-6% after solution, the fixed 0.5-2 of room temperature are removed
Hour.The length of set time is related with the use concentration of trichloroacetic acid, and the concentration used is higher, and protein compression effect is got over
By force, perivitelline increase is more obvious, and the set time can suitably shorten；Conversely, the concentration used is lower, protein compression effect is got over
Weak, perivitelline increase is more unobvious, and the set time can be appropriately extended.According to perivitelline size, the trichloroacetic acid of 6% concentration is most short
Set time is 0.5 hour, and the trichloroacetic acid of the 4% concentration most short set time is 1 hour.
2) paraformaldehyde is fixed: being outwelled solution of trichloroacetic acid, 4% paraformaldehyde solution is added, 4 DEG C of fixations are overnight.
3) fixer is replaced: after paraformaldehyde has been fixed, with phosphate buffer solution (PBS component: 128mM NaCl, 2mM
KCl,8mM NaH2PO4,2mM KH2PO4, pH 7.2) the corresponding 3/4-4/5 volume of displacement paraformaldehyde fixer, and in 4 DEG C
It saves stand-by；Repeated flushing is not needed when with phosphate buffer displacement fixer, not only improves the long-term preservation of sample in this way, again
It will not influence dyeing effect.
4): there is apparent perivitelline between the fertilization membrane and vitellinae membrana of fertilized eggs after fixed in striping, under anatomical lens, uses
Tip tweezers or dissecting needle remove fertilization membrane, obtain complete embryo, are saved with phosphate buffer (PBS) in 4 DEG C, or
100% methanol is used for subsequent whole embryo immunohistochemistry or cytology correlative study in -20 DEG C of freezen protectives.
The present invention is described in detail with reference to the accompanying drawings and examples.
Embodiment 1
The fertilized eggs of turbot after fertilization 20min first fix 2 hours with 4% trichloroacetic acid, then with 4 DEG C of 4% paraformaldehyde
It is fixed to stay overnight.Detailed process is as follows:
(1) trichloroacetic acid is fixed: anisolecithal egg is sucked in centrifuge tube with plastic suction pipe, remove be added after solution 4% three
Chloroacetic acid solution, room temperature fix 2 hours.
(2) paraformaldehyde is fixed: being outwelled solution of trichloroacetic acid, 4% paraformaldehyde solution is added, 4 DEG C of fixations are overnight.
(3) fixer is replaced: after paraformaldehyde has been fixed, with phosphate buffer solution (PBS component: 128mM NaCl, 2mM
KCl,8mM NaH2PO4,2mM KH2PO4, pH 7.2) the corresponding 3/4-4/5 volume of displacement paraformaldehyde fixer, and in 4 DEG C
It saves stand-by.
(4) striping: there is apparent perivitelline (such as Fig. 1 a) in embryo after fixed, by the embryo after striping in phosphate buffer
4 DEG C save stand-by (such as Fig. 1 b).From Fig. 1 a and 1b: embryo's shrinkage after fixed is less, and embryo is complete after striping.
Embodiment 2
The fertilized eggs of turbot after fertilization 20min first fix 0.5 hour with 6% trichloroacetic acid, then with 4% paraformaldehyde 4
It is DEG C fixed overnight.Detailed process is as follows:
(1) trichloroacetic acid is fixed: anisolecithal egg is sucked in centrifuge tube with plastic suction pipe, remove be added after solution 6% three
Chloroacetic acid solution, room temperature fix 0.5 hour.
(2) paraformaldehyde is fixed: being outwelled solution of trichloroacetic acid, 4% paraformaldehyde solution is added, 4 DEG C of fixations are overnight.
(3) it replaces fixer: after paraformaldehyde has been fixed, replacing corresponding 3/4-4/5 volume with phosphate buffer solution (PBS)
Paraformaldehyde fixer, and save in 4 DEG C stand-by.
(4) striping: the perivitelline of embryo is clearly visible (such as Fig. 2 a) after fixed, by the embryo after striping in phosphate buffer
4 DEG C save stand-by (such as Fig. 2 b).From Fig. 2 a and 2b: the perivitelline of embryo is more obvious than embodiment 1 after fixed, embryo after striping
Completely.
Embodiment 3
The fertilized eggs of turbot after fertilization 20min first fix 1 hour with 6% trichloroacetic acid, then with 4 DEG C of 4% paraformaldehyde
It is fixed to stay overnight.Detailed process is as follows:
(1) trichloroacetic acid is fixed: anisolecithal egg is sucked in centrifuge tube with plastic suction pipe, remove be added after solution 6% three
Chloroacetic acid solution, room temperature fix 1 hour.
(2) paraformaldehyde is fixed: being outwelled solution of trichloroacetic acid, 4% paraformaldehyde solution is added, 4 DEG C of fixations are overnight.
(3) it replaces fixer: after paraformaldehyde has been fixed, replacing corresponding 3/4-4/5 volume with phosphate buffer solution (PBS)
Paraformaldehyde fixer, and save in 4 DEG C stand-by.
(4) striping: the perivitelline of embryo is obvious (such as Fig. 3 a) after fixed, by the embryo after striping in 4 DEG C of phosphate buffer
Save stand-by (such as Fig. 3 b).From Fig. 3 a and 3b: the perivitelline of embryo is more more obvious than embodiment 2 after fixed, embryo after striping
Tire is complete.
Claims (7)
1. a kind of whole embryo fixing means of striping anisolecithal egg, which is characterized in that the method comprises the following steps that
1) trichloroacetic acid is fixed: anisolecithal egg being placed in progress room temperature in trichloroacetic acid solution and is fixed；
2) paraformaldehyde is fixed: removing trichloroacetic acid solution, anisolecithal egg is fixed with paraformaldehyde solution again；
3) it replaces fixer: using phosphate buffer solution PBS replacing section paraformaldehyde fixer, 4 DEG C save for use；
4) striping: the fertilization membrane of the fertilized eggs after will be fixed in 3) removes, and complete embryo is obtained, with phosphate buffer or methanol
It saves.
2. the method as described in claim 1, which is characterized in that described 1) described in the concentration of trichloroacetic acid solution be
4-6%.
3. the method as described in claim 1, which is characterized in that described 1) in the room temperature set time be 0.5-2h.
4. the method as described in claim 1, which is characterized in that described 2) in paraformaldehyde solution concentration be 4%.
5. the method as described in claim 1, which is characterized in that described 3) described in phosphate buffer solution PBS component
It is as follows: 128mM NaCl, 2mM KCl, 8mM NaH2PO4,2mM KH2PO4,pH 7.2。
6. the method as described in claim 1, which is characterized in that the method comprises the following steps that
1) trichloroacetic acid is fixed:
Anisolecithal egg is sucked in centrifuge tube, the solution of trichloroacetic acid of addition 4-6% after solution is removed, room temperature is 0.5-2 hours fixed；
2) paraformaldehyde is fixed: removal solution of trichloroacetic acid, 4% paraformaldehyde solution is added in anisolecithal egg, 4 DEG C of fixations are overnight
Or room temperature is 2-3 hours fixed；
3) fixer is replaced: after paraformaldehyde has been fixed, with the paraformaldehyde of phosphate buffer solution PBS displacement 3/4-4/5 volume
Solution, and saved for use in 4 DEG C；
4) striping: the fertilization membrane of step 3) treated fertilized eggs is removed, and obtains fixed complete embryo.
7. method as claimed in claim 6, which is characterized in that described 1) in be to be fixed using the trichloroacetic acid of 6% concentration
The trichloroacetic acid of 0.5 hour or 4% concentration fixes 1 hour.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910679581.9A CN110286019B (en) | 2019-04-26 | 2019-04-26 | Whole embryo fixing method for membrane-removed yellow eggs |
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| CN201910342535.X | 2019-04-26 | ||
| CN201910679581.9A CN110286019B (en) | 2019-04-26 | 2019-04-26 | Whole embryo fixing method for membrane-removed yellow eggs |
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2019
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| CN103674662A (en) * | 2013-11-22 | 2014-03-26 | 大连海洋大学 | DAPI staining method for fish early embryonic development |
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