CN108169474A - A kind of novel cell fixative - Google Patents
A kind of novel cell fixative Download PDFInfo
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- CN108169474A CN108169474A CN201810028580.3A CN201810028580A CN108169474A CN 108169474 A CN108169474 A CN 108169474A CN 201810028580 A CN201810028580 A CN 201810028580A CN 108169474 A CN108169474 A CN 108169474A
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- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
Abstract
The invention belongs to biotechnologies, and in particular to a kind of novel cell fixative.The technical solution adopted is that：Using L ascorbic acid as a kind of novel cell fixative, applied to immunofluorescence technique.The present invention reports L ascorbic acid for the first time cell to be fixed as cell fixative.Form and structure can not only be completely kept through the fixed cell of L ascorbic acid, its antigen form can also be preserved well, is conducive to the carry out specific bond with corresponding antibodies.Meanwhile L ascorbic acid can also carry out cell certain penetrating processing, be not required to additionally add cell-permeant agent when fixing cell.Moreover, L ascorbic acid itself is nontoxic, in soluble easily in water, prepare and simple, quick, environmentally friendly using process, no poisonous and harmful substance generates.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of novel cell fixative.
Background technology
Immunofluorescence technique (Immunofluorescence, IF) is a kind of based on antigen-antibody reaction, with fluorescein mark
The antibody of note tracks the experimental technique of the presence of intracellular specific antigen (generally protein) and distribution, in modern biology
Research field has extensive and important application.When carrying out immunofluorescence experiment, it is necessary first to which the cells are fixed, makes the egg of cell
White matter solidifies, and terminates various enzymatic reactions, prevents aqtocytolysis or broken, in the form of keeping cell original and structure.Therefore,
The fixed key of cell is：Suitable cell fixative is selected, cellular morphology to be kept not change, while holding antigen in situ
Three-dimensional structure, avoid inactivation or the disperse of antigen.
The type of cell fixative is more, and currently used cell fixative mainly has aldehydes, alcohols etc., wherein, poly
Formaldehyde (Paraformaldehyde, PFA) is the most normal in immunofluorescence experiment because of its good cell and antigen stationarity
With.But paraformaldehyde has certain toxicity in itself, and the formaldehyde that when heating generates can cause environmental pollution again, to experimenter
Body damages.Therefore, for immunofluorescence experiment, it is badly in need of the harmless high efficiency cell fixative of exploitation novel non-toxic.
Invention content
The object of the present invention is to provide a kind of novel cell fixatives.
For achieving the above object, the technical solution adopted in the present invention is：A kind of novel cell fixative, effectively
Ingredient is L-AA.
Correspondingly, application of the L-AA in immunofluorescence technique.
Preferably, application of the L-AA in immunofluorescence technique, including as a kind of cell fixative.
Preferably, application of the L-AA in immunofluorescence technique is further included as a kind of cell-permeant agent.
Preferably, the L-AA need to be formulated as solution use, and it is water or PBS buffer solution or life to prepare using solvent
Manage brine.
Preferably, a concentration of 50mM~1M of the L-AA solution.
Preferably, a concentration of 100mM of the L-AA solution.
Preferably, application of the L-AA in immunofluorescence technique, is fixed and penetrating adherent growth is thin
The method of born of the same parents is：Cell climbing sheet is taken, cell is cleaned with PBS；Cell climbing sheet then is impregnated with the L-AA solution of 100mM,
It is placed in room temperature.
Preferably, application of the L-AA in immunofluorescence technique, is fixed and penetrating suspension growth is thin
The method of born of the same parents is：Suspension cell is collected, cell is resuspended with PBS, cell suspension is drawn and is placed on cleaning carrier, 25~45 DEG C certainly
So drying adds the L-AA solution covering cell of 100mM, is placed in room temperature.
Preferably, cell to be fixed need to be completely covered for the L-AA solution of addition.
The invention has the advantages that：
1st, reporting the solution prepared by L-AA (L-ascorbic acid, vitamin C) for the first time can be used as carefully
Cell is fixed in born of the same parents' fixative, and the solvent for preparing solution can be water, phosphate buffer (phosphate buffer
Saline, hereinafter referred to as PBS), physiological saline etc. can dissolve L-AA, it is harmless to cytotoxic and can help to maintain cell
The solution of osmotic pressure.
2nd, its form and structure can not only be completely kept through the fixed cell of L-AA, can also protected well
Antigen is deposited, is conducive to its specific bond with corresponding antibodies.
3rd, L-AA can also improve membrane passage while cell is fixed, and cell is carried out
Certain penetrating processing, without as other regular growth fixatives, additionally adding cell-permeant agent.
4th, common cell fixative, such as paraformaldehyde, itself have certain toxicity, and L-AA to human body without
Toxic side effect, it is environmentally friendly.
5th, common cell fixative, such as paraformaldehyde, solubility in water is not high, and preparation generally requires heating
Stirring heats and such reagent dissociation is often promoted to go out the stronger substance of some toxicity, such as formaldehyde to help to dissolve；This
The L-AA that invention uses is easily soluble in water, and process for preparation is simple, quick, environmental protection, more not poisonous and harmful without heating
Substance generates.
Description of the drawings
Fig. 1 is immunofluorescence technique indirect method schematic diagram；
Fig. 2 is immunofluorescence technique experiment flow schematic diagram；
Fig. 3 is testing result of the different cell fixatives to MES23.5 intracellular tyrosine hydroxylases；
Fig. 4 is the testing result of different cell fixatives intracellular γ-tubulin to NB4；
Fig. 5 is testing result of the different cell fixatives to GFAP in astroglia；
Fig. 6 is testing result of the different cell fixatives to MAP2 in GFAP in astroglia and neuron；
When Fig. 7 is without using cell-permeant agent, different cell fixatives are to the testing result of GFAP in astroglia.
Specific embodiment
Immunofluorescence technique is mainly divided to two kinds of direct method and indirect method, main difference is that antigen whether directly with label
The antibody of fluorescein combines.Generally indirect method uses more, therefore herein using indirect method, concrete principle is as shown in Figure 1, experiment
Flow is as shown in Figure 2.
Following embodiment is intended merely to the application method and technology that those skilled in the art is allowed to understand the present invention in more detail
Effect is limited not as to the protection of the present invention.
Herein approach is bought using reagent：
(1) N1 additives, DMEM/F12medium, RPMI medium 1640, dual anti-(Pen .- Strep), phosphoric acid
Salt buffer (PBS) is purchased from Wisent；
(2) Neurobasal medium, B27 additives, MEM medium are bought from Invitrogen, primary antibody Mouse
It is anti-Glial fibrillary acidic protein (Anti-GFAP), Poly-L-Lysine, L-AA, more
Polyformaldehyde (PFA), 4 ', 6-diamidino-2-phenylindole dihydrochloride (DAPI) are purchased from Sigma；
(3) primary antibody Rabbit anti-Microtubule-associated protein (Anti-MAP2), primary antibody
Rabbit anti-Tyrosine hydroxylase (Anti-TH) are purchased from Millipore；
(4) primary antibody Rabbit anti-γ-tubulin are purchased from Santa Cruz Biotechnology；
(5) fluorescent marker secondary antibody goat anti-rabbit igg (TRITC labels), goat anti-rabbit igg (FITC labels), goat anti-mouse igg
(FITC labels) is purchased from Beijing company of Zhong Shan Golden Bridge；
(6) anti-fluorescence decay mountant is purchased from Beijing Puli's lema gene Technology Co., Ltd.；
(7) fetal calf serum is purchased from Lanzhou lark, and Triton X-100, bovine serum albumin(BSA) (BSA) are purchased from green skies company.
Embodiment one：L-AA is as detection (MES23.5 cell) of the cell fixative to cell line single antigen
The main purpose of immunofluorescence experiment is to detect the presence and its distribution of specific antigen in sample.Therefore the present invention is first
MES23.5 cells have first been selected as sample detection specific antigen therein.MES23.5 cells be rat embryo midbrain cell with
A kind of hybridoma that mouse neuroblastoma-glioma cell line N18TG2 hybrid fusions form, contains into the cell
Abundant tyrosine hydroxylase is the marker of dopaminergic neuron for synthesizing dopamine.
1st, preparing experiment cell
Dopaminergic neuron cell line MES23.5 used in the present invention comes from applicant's Laboratories Accession.
2nd, prepare reagent
DMEM/F12 culture mediums：DMEM/F12medium+5% fetal calf serum+1%N1 additives+1% are dual anti-.
3rd, cell is cultivated
It is inoculated in DMEM/F12 culture mediums, using carbon dioxide incubator in 5%CO2, pasted under conditions of 37 DEG C
Wall culture.
4th, cell climbing sheet is made
Round coverslip is cleaned, is positioned over after high-temperature sterilization in 6 orifice plates.The Poly- of 2mL is added in per hole in 6 orifice plates
L-Lysine solution and do not had coverslip, and placed for 24 hours in 37 DEG C of incubators, make coverslip surface by Poly-L-Lysine packets
Quilt.
Poly-L-Lysine solution is discarded, is cleaned twice with PBS.By MES23.5 cells kind in the 6 orifice plate lids handled well
On slide, when being put into incubator culture to cell and covering with 60% region of coverslip, 6 orifice plates are taken out and carries out cell and fixes.
5th, fixed cell
(1) configuration of cell fixer：
Experimental group：L-AA solid powder is weighed, being dissolved in PBS makes its final concentration of 100mM.
Control group：Paraformaldehyde solid powder is weighed, dissolves by heating and 4% paraformaldehyde (w/v) is obtained in PBS.
(2) cell is fixed：Above-mentioned 6 orifice plates containing cell climbing sheet are taken out into incubator, culture medium is discarded, is cleaned with PBS
Cell 3 times.The L-AA solution (experimental group) or 4% paraformaldehyde solution of 2mL 100mM is added in into 6 orifice plates respectively
(control group) impregnates cell climbing sheet to fix cell, fixes 30min at room temperature.
6th, cell-permeant
After the completion of cell is fixed, the cell fixer in 6 orifice plates is discarded, PBS is added in and is cleaned.By 6 holes during cleaning
Plate is placed in low speed water horizontal tremble in decolorization swinging table and swings, each 5min, cleans 3 times.PBS is discarded, adds in 2mL per hole in 6 orifice plates
0.2%Triton X-100 solution (PBS preparations) carries out cell-permeant, places 30min at room temperature.
7th, sample closing is combined with antibody
Using specific antibody Rabbit Anti-TH as primary antibody, goat anti-rabbit igg (FITC labels) is as secondary antibody, with 2%
BSA (PBS preparations) it is diluted respectively it is spare, diluted concentration refer to used primary antibody and secondary antibody specification, generally
1：200~1：1000, the present embodiment 1:500.
Cell-permeant liquid is discarded, PBS is cleaned 3 times, each 5min.The skim milk of 2mL5% is added in 6 orifice plates per hole
(PBS preparations) carries out sample closing, is placed at room temperature for 1h.After sample closing, primary antibody is placed in corresponding cell at 4 DEG C and is incubated jointly
It educates overnight.6 orifice plates are taken out, discard primary antibody, PBS is cleaned 6 times, each 5min.Secondary antibody (fluorescein label) and cell are placed again
It is protected from light at room temperature and is incubated 3h.Secondary antibody is discarded, PBS is cleaned 6 times, and each 5min is protected from light operation.
8th, core, mounting, detection are contaminated
After the completion of cell is combined with antibody, the DAPI (PBS preparations) of 1 μ g/mL and cell are incubated at room temperature in the place of being protected from light
15min, to be dyed to nucleus.DAPI dyeing liquors are discarded, PBS is cleaned 3 times.The anti-fluorescent quenching mountants of 15 μ L is taken to be added dropwise
In on coverslip cell, finally coverslip is buckled on clean glass slide, completing the preparation of immunofluorescence experiment sample, 4
It DEG C is kept in dark place.Using the sample for preparing of laser confocal microscope (Carl Zeiss) observation, image using ZEN softwares into
Row acquisition and output.
9th, the results are shown in Figure 3, and wherein scale represents 50 μm.DAPI is that a kind of fluorescence that can be combined with DNA strengths contaminates
Material, energy penetration cell film dye nucleus, therefore are commonly used to guide the position of nucleus in immunofluorescence experiment.Junket
The antibody institute specific recognition that propylhomoserin hydroxylase (TH) is marked by fluorescein FITC is special present in MES23.5 cytoplasm endochylemas
Different enzyme.Therefore successfully immunofluorescence experiment not only needs to show tyrosine hydroxylase presence or absence, it is also necessary to show its point
Cloth range.From figure 3, it can be seen that after carrying out cell fixation using 4% paraformaldehyde, the existence position of tyrosine hydroxylase is thin
Born of the same parents' endochylema with the position of nucleus and misaligned (in the Merge of PEF, two arrow indicating positions are misaligned), illustrates more than 4%
Polyformaldehyde can carry out good detection and positioning to the tyrosine hydroxylase antigen in MES23.5 cells.Use L- Vitamin Cs
When acid carries out tyrosine hydroxylase detection, same result can be obtained.This shows L-AA with paraformaldehyde in cell
The preservation of proteantigen three-dimensional structure is had same effect during fixed.Therefore for detecting and positioning cell cytosol
In protein enzyme, L-AA and paraformaldehyde can be used as good cell fixative to be applied to immunofluorescence experiment
In.
Embodiment two：The influence (NB4 cells) that L-AA detects cell line single antigen as cell fixative
Tubulin is the antigen protein often detected in a kind of cytoskeletal protein and immunofluorescence experiment, wherein
The one kind of γ-tubulin as tubulin, is primarily present in the centerbody of cell, and key is played in the assembling of micro-pipe
Effect.The present embodiment selects Human acute promyelocytic leukemia cell strain NB4, and as detection cell, displaying L-AA is made
It is fixative to the detection result of γ-tubulin in NB4 cells.
1st, preparing experiment cell
Human acute promyelocytic leukemia cell strain NB4 used in the present invention comes from applicant's Laboratories Accession.
2nd, prepare reagent
1640 culture mediums：RPMI medium 1640+10% fetal calf serums+1% are dual anti-.
3rd, cell is cultivated
It is inoculated in 1640 culture mediums, using carbon dioxide incubator in 5%CO2, carry out suspension training under conditions of 37 DEG C
It supports.
Second day after cell culture passages 2 times, 5min is centrifuged in 800r/min, cell is collected and carries out cell and fix.
4th, fixed cell
(1) configuration of cell fixer：
Experimental group：L-AA solid powder is weighed, being dissolved in PBS makes its final concentration of 100mM.
Control group：Paraformaldehyde solid powder is weighed, dissolves by heating and 4% paraformaldehyde (w/v) is obtained in PBS.
(2) cell is fixed：The above-mentioned cell being collected by centrifugation is resuspended with PBS, adjustment cell concentration to 1,000,000/mL
Cell suspension is drawn in (coverslip is pre-placed in 6 orifice plates) on clean coverslip in left and right.The lid of cell suspension will be loaded with
Slide is positioned over natural drying in 37 DEG C of baking ovens, adds in the L-AA solution of 2mL 100mM into 6 orifice plates respectively later
(experimental group) or 4% paraformaldehyde solution (control group) impregnate coverslip to fix cell, fix 30min at room temperature.
5th, cell-permeant
After the completion of cell is fixed, the cell fixer in 6 orifice plates is discarded, PBS is added in and is cleaned.By 6 holes during cleaning
Plate is placed in low speed water horizontal tremble in decolorization swinging table and swings, each 5min, cleans 3 times.PBS is discarded, adds in 2mL per hole in 6 orifice plates
0.2%Triton X-100 solution (PBS preparations) carries out cell-permeant, places 30min at room temperature.
6th, sample closing is combined with antibody
Use Rabbit Anti- γ-tubulin (1：400) as primary antibody, using goat anti-rabbit igg (FITC is marked, 1：
100) as secondary antibody.It is diluted respectively with 2%BSA (PBS preparations), it is spare.
Cell-permeant liquid is discarded, PBS is cleaned 3 times, each 5min.The skim milk of 2mL5% is added in 6 orifice plates per hole
(PBS preparations) carries out sample closing, is placed at room temperature for 1h.After sample closing, primary antibody is placed in corresponding cell at 4 DEG C and is incubated jointly
It educates overnight.6 orifice plates are taken out, discard primary antibody, PBS is cleaned 6 times, each 5min.Secondary antibody (fluorescein label) and cell are placed again
It is protected from light at room temperature and is incubated 3h.Secondary antibody is discarded, PBS is cleaned 6 times, and each 5min is protected from light operation.
7th, core, mounting, detection are contaminated
After the completion of cell is combined with antibody, the DAPI (PBS preparations) of 1 μ g/mL and cell are incubated at room temperature in the place of being protected from light
15min, to be dyed to nucleus.DAPI thermocolour liquid is discarded, PBS is cleaned 3 times.The anti-fluorescent quenching mountants of 15 μ L is taken to be added dropwise
In on coverslip cell, finally coverslip is buckled on clean glass slide, completes the preparation of immunofluorescence sample, 4 DEG C are kept away
Light preserves.The sample prepared using laser confocal microscope (Carl Zeiss) observation, image are adopted using ZEN softwares
Collection and output.
8th, the results are shown in Figure 4, and wherein scale represents 50 μm.Either nucleus or γ-tubulin are can be seen that,
Two kinds of cell fixatives do not interfere with detection of the antibody to target.The nucleus of NB4 can be by DAPI dyeing and in irregular
Shape is spherical, and γ-tubulin in cytoplasm due to there is presence, and cytoplasm has wrapped nucleus, therefore Anti-
The position that γ-tubulin antibody is marked is extranuclear cellular portions.DAPI and Anti- γ-tubulin antibody marks
Position should be different, but can the complementary shape into a complete globuli cell.As can be seen from Figure 4, L-AA and more
After polyformaldehyde is fixed, DAPI can dye nucleus, and Anti- γ-tubulin antibody also can be to extranuclear thin
Cytosolic fractions are marked, and the two can overlap the complete NB4 cell shapes of composition.Therefore, L-AA and poly
Formaldehyde can preferably be fixed cell, and not interfere with the detection of this cytoskeletal proteins of γ-tubulin, well
Ground saves the three-dimensional structure and antigen active of cytoskeletal protein.
Embodiment three：Influence (the astroglia that L-AA detects primary cell single antigen as cell fixative
Cell)
Cell category on rat cerebral cortex primary cell creep plate is more, mainly includes：Star horn cell, nerve
Member, oligodendroglia, microglia etc., wherein the form and ratio of various cells have with the difference of incubation time again
Changed.Therefore, if the antigen in specific cells can be detected from this complex sample, is whether to investigate L-AA
Suitable for an important indicator of immunofluorescence experiment.Astroglia is quantity relatively large number of one in cerebral cortex cells
Kind spongiocyte and form of diverse, being capable of specifically expressing glial fibrillary acidic albumen (Glial fibrillary acidic
Protein, GFAP), so GFAP is that common marker that is qualitative and quantitatively detecting is carried out to it.
In order to more fully show L-AA as cell fixative to intracellular single antigen Immunofluorescence test
It influences, the present embodiment has selected rat cerebral cortex primary cell as sample.
1st, preparing experiment cell
Original cuiture rat cerebral cortex star spongiocyte preparation method：Newborn SD rat after taking-up is raw in 12h,
Its brain cortical tissue is taken after the anesthesia of 20% urethane and peels off meninx and blood vessel.It is put into after cortical tissue is shredded containing wood
37 DEG C of digestion 20min in the buffer solution of melon protease and DNase I, later soft piping and druming disperse the cell in cortical tissue
It opens, finally by 200 aim cell screen filtrations of the cortical tissue's suspension dispelled, filtrate is the original for including star spongiocyte
For cerebral cortex cells suspension.
2nd, prepare reagent
The preparation method of Neurobasal culture mediums：Neurobasal medium and MEMmedium are pressed 1：1 mixing, then
It is dual anti-to add 5% fetal calf serum, 1%B27 additives, 0.5% Sodium Pyruvate, 0.5% glucose and 1%.
3rd, cell is cultivated
It is inoculated in Neurobasal culture mediums, is carried out under conditions of 5%CO2,37 DEG C using carbon dioxide incubator
Adhere-wall culture.
4th, cell climbing sheet is made
Round coverslip is cleaned, is positioned over after high-temperature sterilization in 6 orifice plates.The Poly- of 2mL is added in per hole in 6 orifice plates
L-Lysine solution and do not had coverslip, and placed for 24 hours in 37 DEG C of incubators, make coverslip surface by Poly-L-Lysine packets
Quilt.
Poly-L-Lysine solution is discarded, is cleaned twice with PBS.By MES23.5 cells kind in the 6 orifice plate lids handled well
On slide, when being put into incubator culture to cell and covering with 60% region of coverslip, 6 orifice plates are taken out and carries out cell and fixes.
5th, fixed cell
(1) configuration of cell fixer：
Experimental group：L-AA solid powder is weighed, being dissolved in PBS makes its final concentration of 100mM.
Control group：Paraformaldehyde solid powder is weighed, dissolves by heating and 4% paraformaldehyde (w/v) is obtained in PBS.
(2) cell is fixed：Above-mentioned 6 orifice plates containing cell climbing sheet are taken out into incubator, culture medium is discarded, is cleaned with PBS
Cell 3 times.The L-AA solution (experimental group) or 4% paraformaldehyde solution of 2mL 100mM is added in into 6 orifice plates respectively
(control group) impregnates cell climbing sheet to fix cell, fixes 30min at room temperature.
6th, cell-permeant
After the completion of cell is fixed, the cell fixer in 6 orifice plates is discarded, PBS is added in and is cleaned.By 6 holes during cleaning
Plate is placed in low speed water horizontal tremble in decolorization swinging table and swings, each 5min, cleans 3 times.PBS is discarded, adds in 2mL per hole in 6 orifice plates
0.2%Triton X-100 solution (PBS preparations) carries out cell-permeant, places 30min at room temperature.
7th, sample closing is combined with antibody
Use Mouse Anti-GFAP (1：400) as primary antibody, using goat anti-mouse igg (FITC is marked, 1：100) it does
For secondary antibody, primary antibody and secondary antibody are diluted respectively with 2%BSA (PBS preparations).
Cell-permeant liquid is discarded, PBS is cleaned 3 times, each 5min.The skim milk of 2mL5% is added in 6 orifice plates per hole
(PBS preparations) carries out sample closing, is placed at room temperature for 1h.After sample closing, primary antibody is placed in corresponding cell at 4 DEG C and is incubated jointly
It educates overnight.6 orifice plates are taken out, discard primary antibody, PBS is cleaned 6 times, each 5min.Secondary antibody (fluorescein label) and cell are placed again
It is protected from light at room temperature and is incubated 3h.Secondary antibody is discarded, PBS is cleaned 6 times, and each 5min is protected from light operation.
8th, core, mounting, detection are contaminated
After the completion of cell is combined with antibody, the DAPI (PBS preparations) of 1 μ g/mL and cell are incubated at room temperature in the place of being protected from light
15min, to be dyed to nucleus.DAPI thermocolour liquid is discarded, PBS is cleaned 3 times.The anti-fluorescent quenching mountants of 15 μ L is taken to be added dropwise
In on coverslip cell, finally coverslip is buckled on clean glass slide, completes the preparation of immunofluorescence sample, 4 DEG C are kept away
Light preserves.The sample prepared using laser confocal microscope (Carl Zeiss) observation, image are adopted using ZEN softwares
Collection and output.
9th, the results are shown in Figure 5, and wherein scale represents 50 μm.Because DAPI has no cell-specific to nuclear targeting,
So all nucleus in Rat cortical cell are marked by DAPI, in spherical；And astroglia is because of specifically expressing
GFAP and be labeled, in star or radial.GFAP is a kind of albumen for being present in astroglia matter position, therefore immune
In fluorescence photo, it should be located in cytoplasm rather than nucleus.Fig. 5 is shown, uses the cell sample of two kinds of different fixatives
Product, DAPI and the position that GFAP antibody is marked are misaligned (in figure shown in grey arrow).In addition, it is removed in Rat cortical cell
Outside star horn cell, also there are other types of cell, therefore the cell nuclei being colored should theoretically be more than star
The number of horn cell.It is the other cell types of non-astroglia detected shown in white arrow, due to DAPI
To the label of nucleus without specificity, and GFAP antigens have specificity, therefore cell shown in white arrow has only been detected carefully
Karyon.The above results show L-AA as a kind of cell fixative suitable for primary cell the detection of specific antigen and
Positioning.
Example IV：Influence of the L-AA as cell fixative dual anti-former detection to cell
Above-described embodiment illustrates that L-AA is still thin in original cuiture in cell line as cell fixative
Antigen three-dimensional structure can be preferably maintained in the immunofluorescence experiment of born of the same parents, so as to be identified and be examined by specific antibody
It surveys.But it is frequently necessary to even a variety of antigens to two kinds in immunofluorescence experiment to carry out while detect, and must not produce between each other
Raw interference.Preferably to show L-AA in immunofluorescence experiment to influence (double marks of two kinds of antigens while detection
Note), the present embodiment selects rat primary cortex cell as sample, after L-AA is fixed while detects astroglia
With two kinds of differential proteins in neuron.
Microtubule associated protein 2 (Microtubule-associated protein 2, MAP2) special table in neuron
It reaches, the marker of mature neuron is used as in biological study, therefore selection MAP2 albumen is made in the present embodiment
Specific antigen for labeled neurons.
1st, preparing experiment cell
Original cuiture rat cerebral cortex star spongiocyte preparation method：Newborn SD rat after taking-up is raw in 12h,
Its brain cortical tissue is taken after the anesthesia of 20% urethane and peels off meninx and blood vessel.It is put into after cortical tissue is shredded containing wood
37 DEG C of digestion 20min in the buffer solution of melon protease and DNase I, later soft piping and druming disperse the cell in cortical tissue
It opens, finally by 200 aim cell screen filtrations of the cortical tissue's suspension dispelled, filtrate is comprising star spongiocyte and god
Primary cerebral cortex cells suspension through a variety of brain cells such as member.
2nd, cell is cultivated
By above-mentioned cell suspension inoculation in Neurobasal culture mediums, using carbon dioxide incubator in 5%CO2,37
Adhere-wall culture is carried out under conditions of DEG C.
3rd, cell climbing sheet is made
Round coverslip is cleaned, is positioned over after high-temperature sterilization in 6 orifice plates.The Poly- of 2mL is added in per hole in 6 orifice plates
L-Lysine solution and do not had coverslip, and placed for 24 hours in 37 DEG C of incubators, make coverslip surface by Poly-L-Lysine packets
Quilt.
Poly-L-Lysine solution is discarded, is cleaned twice with PBS.By MES23.5 cells kind in the 6 orifice plate lids handled well
On slide, when being put into incubator culture to cell and covering with 60% region of coverslip, 6 orifice plates are taken out and carries out cell and fixes.
5th, fixed cell
(1) configuration of cell fixer：
Experimental group：L-AA solid powder is weighed, being dissolved in PBS makes its final concentration of 100mM.
Control group：Paraformaldehyde solid powder is weighed, dissolves by heating and 4% paraformaldehyde (w/v) is obtained in PBS.
(2) cell is fixed：Above-mentioned 6 orifice plates containing cell climbing sheet are taken out into incubator, culture medium is discarded, is cleaned with PBS
Cell 3 times.The L-AA solution (experimental group) or 4% paraformaldehyde solution of 2mL 100mM is added in into 6 orifice plates respectively
(control group) impregnates cell climbing sheet to fix cell, fixes 30min at room temperature.
6th, cell-permeant
After the completion of cell is fixed, the cell fixer in 6 orifice plates is discarded, PBS is added in and is cleaned.By 6 holes during cleaning
Plate is placed in low speed water horizontal tremble in decolorization swinging table and swings, each 5min, cleans 3 times.PBS is discarded, adds in 2mL per hole in 6 orifice plates
0.2%Triton X-100 solution (PBS preparations) carries out cell-permeant, places 30min at room temperature.
7th, sample closing is combined with antibody
Prepare two kinds of primary antibodies.Primary antibody (1)：Mouse Anti-GFAP(1：400)；Primary antibody (2)：Rabbit Anti-MAP2
(1：500).It is diluted with 2%BSA (PBS preparations).
Prepare two kinds of secondary antibodies.Secondary antibody (1)：Goat anti-rabbit igg (TRITC is marked, and 1：100)；Goat-anti (2)：Mouse IgG (FITC
Label, 1：100).It is diluted with 2%BSA (PBS preparations).
Cell-permeant liquid is discarded, PBS is cleaned 3 times, each 5min.The skim milk of 2mL5% is added in 6 orifice plates per hole
(PBS preparations) carries out sample closing, is placed at room temperature for 1h.
After sample closing, two kinds of primary antibodies are placed in corresponding cell at 4 DEG C and are incubated overnight jointly.6 orifice plates are taken out, are discarded
Primary antibody, PBS are cleaned 6 times, each 5min.Two kinds of secondary antibodies (fluorescein label) and cell placement are protected from light incubation at room temperature again
3h.Secondary antibody is discarded, PBS is cleaned 6 times, and each 5min is protected from light operation.
8th, core, mounting, detection are contaminated
After the completion of cell is combined with antibody, the DAPI (PBS preparations) of 1 μ g/mL and cell are incubated at room temperature in the place of being protected from light
15min, to be dyed to nucleus.DAPI thermocolour liquid is discarded, PBS is cleaned 3 times.The anti-fluorescent quenching mountants of 15 μ L is taken to be added dropwise
In on coverslip cell, finally coverslip is buckled on clean glass slide, completes the preparation of immunofluorescence sample, 4 DEG C are kept away
Light preserves.The sample prepared using laser confocal microscope (Carl Zeiss) observation, image are adopted using ZEN softwares
Collection and output.
9th, the results are shown in Figure 6, and wherein scale represents 50 μm.As can be seen that the GFAP in astroglia is due to quilt
The antibody specific recognition of FITC labels is in star or radial under laser confocal microscope；MAP2 in neuron then by
The antibody specific recognition of TRITC labels, cell are in the distinctive long more dendron shapes of aixs cylinder of neuron；All cells is thin in sample
Karyon is then by DAPI labeled as near spherical.In experiment, the astroglia in same sample is with neuron simultaneously by respectively glimmering
The phenomenon that specific antibody of signal detects and positions, do not interfere with each other, the god marked as shown by arrows for Anti-MAP2
Through first protrusion, it is only located in neuronal cell.The same sample that different antibodies are marked using laser confocal microscope
It takes pictures respectively, after three obtained width pictures carry out Merge, it is found that nucleus, astroglia, neuron can be same
It is significantly distinguished in a sample.Therefore, L-AA is completely suitable for the immunofluorescence of double labelling as cell fixative
Experiment.
Embodiment five：Influence when L-AA fixes cell to cell permeability
As shown in Fig. 2, in conventional immunofluorescence experiment, cell needs to carry out cell penetrating after fixative is fixed
Processing, i.e., punched on cell membrane, is combined into the cell with antigen so that the antibody and fluorescent dye that are added in enter.And
When L-AA is as cell fixative, itself has the function of cell-permeant, without additionally carrying out penetrating processing to cell.
For influence of the displaying L-AA to permeability of cell membrane, the present embodiment is using the Rat Astroglia of long-term cultivation as material
Material compares the effect of L-AA and paraformaldehyde.
1st, preparing experiment cell
The Rat Astroglia preparation method of long-term cultivation：The SD rat children mouses in newborn 12 hours are taken, at anesthesia
After death, young rat cerebral cortex is taken, resolving into unicellular be placed in DMEM/F12 culture mediums with pancreatin cultivates.Treat that cell covers with training
After supporting ware bottom, astroglia is enriched in culture dish by repeatedly passing on.
2nd, cell is handled
Using the method for example IV, cell is cultivated, and it is fixed, but cell-permeant agent is not used in this example
Triton X-100 carry out penetrating processing to cell.Wherein, DAPI can be by complete cell membrane, therefore it enters nucleus
And the process dyed does not need to be penetrating to cell membrane progress.But GFAP is present in the cytoplasm of astroglia, often
In the immunofluorescence experiment of rule, need that cell membrane is carried out penetrating processing in advance, the primary antibody and secondary antibody of Anti-GFAP could enter
Cell marks GFAP.
3rd, using the method for example IV, dye core, mounting, detection are carried out.
4th, the results are shown in Figure 7.In the case of unused cell-permeant agent Triton X-100, cell is through paraformaldehyde
After fixation, final laser co-focusing photo can not carry out the GFAP albumen in primary astroglial cells clear effective inspection
It surveys.This shows after fixing cell using paraformaldehyde that the integrality of cell membrane is not destroyed, the antibody and fluorescein added in
Cell and antigen binding are cannot be introduced into, therefore laser confocal microscope can not detect GFAP albumen.Equally it is being not used carefully
In the case of penetrating dose of born of the same parents, for cell after L-AA is fixed, laser confocal microscope can clearly show that star
The GFAP of spongiocyte.And the plasm where GFAP and nucleus that DAPI is dyed are in distribution and misaligned, and
It is that common complementation shows an intact cell, this is consistent with theoretic situation.This shows using L-AA right
While cell is fixed, can to a certain extent destroy cell membrane integrality, enable antibody enter cell interior with
Antigen-specific combines.
Above-mentioned phenomenon shows that L-AA is tested as cell fixative for cellular immunofluorescence, can not only fix
Cell goes back permeable cell film, can substitute paraformaldehyde in conventional method (cell fixative) simultaneously and Triton X-100 are (thin
Penetrating dose of born of the same parents) two kinds of reagents.
Therefore, it when carrying out immunofluorescence experiment using L-AA as cell fixative, does not need to additionally use again
Cell-permeant agent.
Embodiment six：Different solvents dissolve L-AA, fixed and permeabilized cells effects
1st, by the method for embodiment five, prepare reagent, make 3 groups of cell climbing sheet.
2nd, fixed cell
(1) cell fixer, penetrating dose of configuration：
The L-AA solid powder of equivalent is weighed, is divided into 3 groups, is dissolved separately in PBS, water and physiological saline, makes
The final concentration of 100mM of each group.
(2) according to the method for embodiment five, cell is fixed respectively, without penetrating processing.
3rd, by the method for embodiment five, dye core, mounting, detection are carried out respectively to each group cell.
4th, each group result is suitable with the result of Fig. 7, and the L-AA solvent prepared using different solvents consolidates cell
It is set for, without influence, realizing goal of the invention with permeation.The result shows that it is glimmering that L-AA is applied to cellular immunity
When light is tested, selected solvent is every to dissolve L-AA almost without influence, and it is harmless to treat fixed cytotoxic,
The reagent of its survival is not influenced, can be used.
Claims (10)
1. a kind of novel cell fixative, which is characterized in that active ingredient is L-AA.
Application of the 2.L- ascorbic acid in immunofluorescence technique.
3. application of the L-AA as claimed in claim 2 in immunofluorescence technique, it is characterised in that：The application packet
It includes as a kind of cell fixative.
4. application of the L-AA as claimed in claim 2 in immunofluorescence technique, it is characterised in that：The application is also
Including as a kind of cell-permeant agent.
5. application of the L-AA as claimed in claim 2 in immunofluorescence technique, it is characterised in that：The L- is anti-bad
Hematic acid need to be formulated as solution use, and it is water or PBS buffer solution or physiological saline to prepare using solvent.
6. application of the L-AA as claimed in claim 5 in immunofluorescence technique, it is characterised in that：The L- is anti-bad
A concentration of 50mM~1M of hematic acid solution.
7. application of the L-AA as claimed in claim 6 in immunofluorescence technique, it is characterised in that：The L- is anti-bad
A concentration of 100mM of hematic acid solution.
8. application of the L-AA as claimed in claim 7 in immunofluorescence technique, it is characterised in that：It is fixed and penetrating
The method of adherent growth cell is：Cell climbing sheet is taken, cell is cleaned with PBS；Then impregnated with the L-AA solution of 100mM
Cell climbing sheet is placed in room temperature.
9. application of the L-AA as claimed in claim 7 in immunofluorescence technique, it is characterised in that：It is fixed and penetrating
The method of suspension growth cell is：Suspension cell is collected, cell is resuspended with PBS, cell suspension is drawn and is placed on cleaning carrier,
25~45 DEG C of natural dryings add the L-AA solution covering cell of 100mM, are placed in room temperature.
10. application of the L-AA as claimed in claim 8 or 9 in immunofluorescence technique, it is characterised in that：It adds in
Cell to be fixed need to be completely covered for L-AA solution.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113740522A (en) * | 2021-09-01 | 2021-12-03 | 陕西脉元生物科技有限公司 | Antigen repairing liquid, cell climbing sheet prepared by using antigen repairing liquid, and preparation method and application of cell climbing sheet |
| CN114258909A (en) * | 2021-12-14 | 2022-04-01 | 苏州良辰生物医药科技有限公司 | Cell fixing agent and cell fixing method |
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| CN1358866A (en) * | 2001-12-12 | 2002-07-17 | 李瑞峰 | Novel technology of peripheral blood white cell hybridization in situ experiment |
| CN102313801A (en) * | 2011-06-03 | 2012-01-11 | 南开大学 | Metabolic labeling method of antibody and application of antibody in fluorescence detection |
| CN105866432A (en) * | 2016-05-11 | 2016-08-17 | 天津普瑞赛尔生物科技有限公司 | Kit for identifying dental pulp mesenchymal stem cells |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1358866A (en) * | 2001-12-12 | 2002-07-17 | 李瑞峰 | Novel technology of peripheral blood white cell hybridization in situ experiment |
| CN102313801A (en) * | 2011-06-03 | 2012-01-11 | 南开大学 | Metabolic labeling method of antibody and application of antibody in fluorescence detection |
| CN105866432A (en) * | 2016-05-11 | 2016-08-17 | 天津普瑞赛尔生物科技有限公司 | Kit for identifying dental pulp mesenchymal stem cells |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113740522A (en) * | 2021-09-01 | 2021-12-03 | 陕西脉元生物科技有限公司 | Antigen repairing liquid, cell climbing sheet prepared by using antigen repairing liquid, and preparation method and application of cell climbing sheet |
| CN114258909A (en) * | 2021-12-14 | 2022-04-01 | 苏州良辰生物医药科技有限公司 | Cell fixing agent and cell fixing method |
| CN114258909B (en) * | 2021-12-14 | 2023-09-22 | 苏州良辰生物医药科技有限公司 | Cell fixing agent and cell fixing method |
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