CN110132672A

CN110132672A - A kind of histocyte fixating reagent and histocyte fixing means

Info

Publication number: CN110132672A
Application number: CN201910404527.3A
Authority: CN
Inventors: 王樱达; 王郑; 朱晓璐; 王琳琳; 刘倩; 陈建树
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-05-16
Filing date: 2019-05-16
Publication date: 2019-08-16
Anticipated expiration: 2039-05-16
Also published as: CN110132672B

Abstract

The invention discloses a kind of histocyte fixating reagent and histocyte fixing means, the effective component of the cell fixating reagent is C₁₆H₂₈O₄N₃.The step of histocyte fixing means, is as follows: (1) appropriate PBS buffer solution being added, in culture medium for histiocyte；(2), configured cell fixating reagent as claimed in claim 2 is added in PBS buffer solution, the cell fixating reagent and the Volume fraction of PBS buffer solution are 1%-20%, it is 0.5-4 hours fixed, cell fixating reagent toxicity of the invention is low, non-volatility, it is preferable to histiocytic fixed effect, 6 months or more fixed effects can be maintained in PBS buffer solution.For fixing means provided by the invention compared with other cell fixation methods, outstanding advantages are that ionic liquid is non-volatile, are safe from harm to human body, in addition have easy to operate, it is at low cost, it is fixed after cell can long-term preservation in PBS aqueous solution, and cell fixed effect is preferable.

Description

A kind of histocyte fixating reagent and histocyte fixing means

Technical field

The present invention relates to a kind of histocyte fixating reagent and histocyte fixing means, belong to field of biotechnology.

Background technique

The type of histocyte fixative is more, and currently used cell fixative mainly has: aldehydes (common formaldehyde, penta Dialdehyde and paraformaldehyde), alcohols (common ethyl alcohol), other (acetone).

Aldehydes:

Formaldehyde (formalin) is most widely used, principle: forming intermolecular crosslinking, influences protein configurations and be allowed to fixed. Advantage: morphosis is kept, and penetrability is strong, and tissue contracts are few.Disadvantage: it is formic acid that formaldehyde, which is placed oxidable too long, is made molten Liquid PH is reduced, and influences to dye；The amino of aldehyde radical and antigen protein is cross-linked to form carboxymethyl, goes out the three-dimensional conformation of antigenic determinant Existing spatial obstacle；The network that intermolecular cross-linking is formed may partially or completely cover certain antigenic determinants, and being allowed to cannot Sufficiently exposure.It can cause the coloration result of false negative.

Glutaraldehyde, penetrability is strong, and fine structure is kept, but fights original certain influence, often combines with other fixatives As immuno-electron microscope fixer.

Paraformaldehyde (common 4%), can be used for immuno-electron microscope；It can also be used for immunofluorescence dyeing.It predominantly detects in tissue The delicate antigen of some performances especially cell surface antigen, such as all kinds of lymphocytes break up determinant (CD), Main Tissues Compatibility antigen etc..

Alcohols:

Most common alcohols fixative is ethyl alcohol.Its fixed function: precipitate intracellular protein, carbohydrate.Advantage: it wears Permeability is strong, antigenicity is kept.Disadvantage: dehydration property is strong, and Yi Yinqi tissue contracts are hardened, and influences chipping qualities, thus ethyl alcohol is solid Fix time unsuitable too long (in 2h).Ethyl alcohol keeps protein-denatured effect light, can be redissolved after fixed；In dyeing course, when incubation Between it is long, antigen can be lost and weaken response intensity.

Other fixatives:

Acetone is kept to antigenic, but dehydration property is stronger, less to be used for tissue specimen, but cell climbing sheet common third Ketone is fixed.

Above-mentioned cell fixer is all traditional volatile solvent, also there is certain harm to human body.

Summary of the invention

In order to overcome above-mentioned deficiency, the present invention provides a kind of new histocyte fixating reagents and histocyte fixation side Method employs ionic liquids to replace traditional cell fixative.

Ionic liquid, usually also referred to as ionic liquid at room temperature, are DEG C salt being in a liquid state under room temperature or low temperature.It is generally by body Relatively large, the asymmetric organic cation of product and the relatively small inorganic anion of volume are formed, due to zwitterion number Mesh is equal, thus shows electroneutral on the whole.Ionic liquid has excellent physicochemical properties and can modify, the yin-yang of modulation Ionic structure.Using ionic liquid as the solvent of reaction system or catalyst, since no vapour pressure, liquid temperature range are wide, point It from being easy to carry out, has that homogeneous catalysis is high-efficient, the segregative advantage of heterogeneous catalysis concurrently, and can be recycled, receive in recent years The extensive concern of each field scholar.

Technical scheme is as follows:

A kind of histocyte fixating reagent, the cell fixating reagent is a kind of ionic liquid, and effective component is C₁₆H₂₈O₄N₃, chemical structural formula are as follows:

The cell fixative is C₁₆H₂₈O₄N₃With the mixed liquor of water.

C₁₆H₂₈O₄N₃Volume fraction with the mixed liquor of water is 1%-20%.Preferred volume score ratio is 10%.

A kind of histocyte fixing means, described steps are as follows:

(1), appropriate PBS buffer solution is added in culture medium for histiocyte；

(2), configured cell fixating reagent as claimed in claim 2 is added in PBS buffer solution, the cell is solid The Volume fraction for determining reagent and PBS buffer solution is 1%-20%, preferably 10%；It is 0.5-4 hours, preferably 3 hours fixed.

Advantageous effects of the invention:

Ionic liquid provided by the invention have catalytic performance is high, easily separate with product, Yi Huishou, it is reusable, make The advantages that with facilitating is the ideal substitute of traditional volatile solvent and catalyst, oneself is through becoming a kind of green material or Jie Matter provides new opportunity for new " soft " functional material of materialized scholar development, while also finding a kind of green for chemist Medium provide new thinking.

Moreover, ionic liquid toxicity of the invention is low, non-volatility is preferable to histiocytic fixed effect, PBS buffering 6 months or more fixed effects can be maintained in liquid.

For fixing means provided by the invention compared with other cell fixation methods, outstanding advantages are that ionic liquid is not waved Hair, is safe from harm to human body, in addition have it is easy to operate, it is at low cost, it is fixed after cell can long-term preservation in PBS aqueous solution, And cell fixed effect is preferable.

Detailed description of the invention

Fig. 1 is that mouse C2C12 cell initially fixes AO-EB colored graph after C2C12 cell；

Fig. 2 is the colored graph of AO-EB after mouse C2C12 cell is fixed 6 months；

Fig. 3 is the bright dark field plot one after mouse C2C12 cell is fixed 6 months；

Fig. 4 is the bright dark field plot two after mouse C2C12 cell is fixed 6 months.

Specific embodiment

The invention will be further described below in conjunction with the accompanying drawings.Following embodiment is only used for clearly illustrating the present invention Technical solution, and not intended to limit the protection scope of the present invention.

The cell fixative is C₁₆H₂₈O₄N₃With the mixed liquor of water.

A kind of histocyte fixing means, described steps are as follows:

(2), configured cell fixating reagent as claimed in claim 2 is added in PBS buffer solution, the cell is solid The Volume fraction for determining reagent and PBS buffer solution is 1%-20%；It is preferred that 10%；It is 0.5-4 hours, preferably 3 hours fixed.

Embodiment:

It chooses mouse cell (C2C12), culture medium therein is sucked out, add suitable PBS buffer solution, institute of the present invention is added The ionic liquid stated makes the volume fraction 10% of PBS intermediate ion liquid, and the set time is 3 hours, and the colored graph of AO-EB is such as Shown in Fig. 1.After fixation, the PBS buffer solution containing ionic liquid is sucked out, then cell 3-5 points after fixing are impregnated with PBS buffer solution Clock, so far, cell is fixed to be completed, and can continue to dye in next step.It is illustrated in figure 2 AO-EB after C2C12 cell is fixed 6 months Colored graph.Such as Fig. 3 and being illustrated in figure 4 the bright dark field plot after C2C12 cell is fixed 6 months.

From Fig. 3 and Fig. 4, we can observe that, after cell is fixed by ionic liquid, form is complete, and it is full, and not It was found that the destruction of apparent cell appearance shrinkage and cell appearance.

We can observe that, karyomorphism is completely full from Fig. 2, and cytoplasm retains completely, and even dyeing.

It may be seen that cell fixed effect is relatively good from implementation, and the cell holding time is also longer.The above institute Stating is only the preferred embodiment of the present invention, it is noted that for those skilled in the art, is not being departed from Under the premise of the technology of the present invention principle, several improvement and deformations can also be made, these improvement and deformations also should be regarded as the present invention Protection scope.

Claims

1. a kind of histocyte fixating reagent, which is characterized in that the effective component of the cell fixating reagent is C₁₆H₂₈O₄N₃, change Learn structural formula are as follows:

2. a kind of histocyte fixating reagent as described in claim 1, it is characterised in that: the cell fixative is C₁₆H₂₈O₄N₃With the mixed liquor of water.

3. a kind of histocyte fixating reagent as claimed in claim 2, it is characterised in that: the C₁₆H₂₈O₄N₃With the mixing of water The volume ratio of liquid is 1%-20%.

4. a kind of histocyte fixating reagent as claimed in claim 3, it is characterised in that: the C₁₆H₂₈O₄N₃With mixing for pure water The preferred volume score ratio for closing liquid is 10%.

5. a kind of histocyte fixing means, it is characterised in that described steps are as follows:

(2), configured cell fixating reagent as claimed in claim 2 is added in PBS buffer solution, the fixed examination of the cell The Volume fraction of agent and PBS buffer solution is 1%-20%；It is 0.5-4 hours fixed.

6. a kind of histocyte fixing means as claimed in claim 5, it is characterised in that the fixed examination of cell in the step (2) The preferred volume score ratio of agent and PBS buffer solution is 10%.

7. a kind of histocyte fixing means as claimed in claim 5, it is characterised in that when cell is fixed in the step (2) Between preferably 3 hours.