CN107703292B - The modification method of BrdU labelled immune fluorescence detection cell Proliferation - Google Patents
The modification method of BrdU labelled immune fluorescence detection cell Proliferation Download PDFInfo
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Classifications
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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
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
-
- 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
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- 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
- G01N33/532—Production of labelled immunochemicals
-
- 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
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
Abstract
The invention discloses a kind of modification methods of Immunofluorescence test BrdU label proliferative cell.Denaturation temperature is fixed on 4 DEG C, renaturation is used uniformly 0.01M Tris-HCL processing, carries out single mark, double marks and three mark dyeing later, and be compared with conventional method.The method of the present invention has extraordinary dyeing effect as the result is shown, and success rate is high, reproducible, easy to operate and saving experimental period.
Description
Technical field
The present invention relates to biological field, in particular to Immunofluorescence test BrdU marks the modification method of proliferative cell.
Background technique
5- bromodeoxyuridine nucleosides (BrdU) is artificial synthesized thymidine analog, synthesizes phase (S phase) in DNA,
BrdU can replace thymidine and penetrate into the DNA molecular replicated, and exist in histocyte without endogenous BrdU.Carefully
BrdU is added in born of the same parents' culture or Active MnO2, uses immunofluorescence dyeing later, BrdU positive cell is proliferative cell.It ties simultaneously
Close other cell markers, double staining can the type of qualitative and quantitative analysis proliferative cell, growth rate and increment cell
Differentiation, migration and survival, it is significant to the research of cyto-dynamics.The side of BrdU labelled immune fluorescence detection cell proliferation
Method is widely used in the research of various cell Proliferations.
The colouring method of BrdU labelled immune fluorescence detection cell Proliferation is varied at present, no unified step.Here is
The step of two kinds of common colouring methods.
Steps are as follows for the first colouring method:
A, rupture of membranes: taking histotomy, and 0.4%triton is added to react at room temperature 20 minutes；
B, repair: by step a, treated that histotomy PBS is washed 5 minutes/3 times, with 0.01mmlol/L citrate
Progress microwave thermal reparation, high fire 5 minutes, small fire 20 minutes, to natural cooling about 1h；
C, be denaturalized: by step b, treated that histotomy PBS is washed 5 minutes/3 times, and 2MHCL is added to act on 30 at 37 DEG C
Minute；
D, renaturation: by step c treated histotomy PBS washes 5 minutes/3 times, then plus the room 0.01M Tris-HCL
Temperature lower reaction 10 minutes；
E, close: by step d, treated that histotomy PBS is washed 5 minutes/3 times, and lowlenthal serum is closed 2 hours；
F, incubate primary antibody: by step e, treated that histotomy gets rid of extra lowlenthal serum, does not wash, and adds BrdU and it is double
4 DEG C of labeling antibody overnight incubations；
G, it incubates secondary antibody: by step f treated 37 DEG C of histotomy rewarming 1 hour, adding 37 DEG C of secondary antibody to be incubated for 1 hour；
H, check: by step g, treated that histotomy PBS is washed 5 minutes/3 times, and DAPI redyes karyon 5-8 minutes；
I, mounting: by step h, treated that histotomy PBS washes 5 minutes/3 times, glycerol mounting.
Steps are as follows for second of colouring method:
A, it is denaturalized: taking histotomy, add 1M HCL to be incubated for 10 minutes on ice, then plus 2M HCL is incubated for 10 at room temperature
Minute, it is acted on 20 minutes at 37 DEG C；
B, renaturation: by step a, treated that histotomy adds 0.1M borate buffer to neutralize at room temperature 10 minutes；
C, rupture of membranes: taking histotomy, and 0.4%triton is added to react at room temperature 20 minutes；
D, repair: by step a, treated that histotomy PBS is washed 5 minutes/3 times, with 0.01mmlol/L citrate
Progress microwave thermal reparation, high fire 5 minutes, small fire 20 minutes, to natural cooling about 1h；
E, close: by step d, treated that histotomy PBS is washed 5 minutes/3 times, and lowlenthal serum is closed 2 hours；
F, incubate primary antibody: by step e, treated that histotomy gets rid of extra lowlenthal serum, does not wash, and adds BrdU and it is double
4 DEG C of labeling antibody overnight incubations；
G, it incubates secondary antibody: by step f treated 37 DEG C of histotomy rewarming 1 hour, adding 37 DEG C of secondary antibody to be incubated for 1 hour；
H, check: by step g, treated that histotomy PBS is washed 5 minutes/3 times, and DAPI redyes karyon 5-8 minutes；
I, mounting: by step h, treated that histotomy PBS washes 5 minutes/3 times, glycerol mounting.Both the above method
For currently used immunofluorescence dyeing method, have following several respects insufficient:
1. denaturation temperature is not fixed.First method directlys adopt 37 ° of denaturation, and BrdU positive mark's location ambiguity is unclear
Clear, second marker cannot normally develop the color when double marks dye, and DAPI or PI dyeing cannot clearly show nucleus.Second of side
Method is denaturalized using 10 minutes → room temperature, 10 minutes → 37 DEG C 20 minutes processing cell or tissues on ice, and operating process is complicated,
" room temperature " is influenced by extraneous factor, such as: winter is different with summer room temperature, thus influences coloration result.It is tested before us
Demonstrating winter room temperature, to make result preferable, and summer room temperature is difficult to catch when DAPI contaminates core as the result is shown.
2. refolding method is different.First method uses 0.01M Tris-HCL renaturation, and second method uses 0.1M boron
Sour renaturation.There are also methods using not renaturation.Our experimental verification is best using 0.01M Tris-HCL renaturation effect.
3. complex steps and disunity.
4. above two conventional method keeps the mono- mark dyeing effect of BrdU very poor, double marks and nuclear staining can not be implemented, serious shadow
Experimental result is rung, therefore, it is necessary to improve to the method.
Summary of the invention:
In view of this, the purpose of the present invention is to provide a kind of BrdU labelled immune fluorescent staining of improvement detection cells to increase
The method grown can carry out single mark, double marks, three mark dyeing, and dyeing effect is clear, and repeatability is high, and method is easy, economical, controllably
Property is strong, and unfavorable factor is few.
The method and step for the BrdU labelled immune fluorescent staining detection cell Proliferation that the present invention improves is as follows:
A, rupture of membranes: taking histotomy to dry in the air at room temperature 15 minutes, and then plus 0.4%triton reacts 20 minutes at room temperature；
B, repair: by step a, treated that histotomy PBS is washed 5 minutes/3 times, with 0.01mmlol/L citrate
Progress microwave thermal reparation, high fire 5 minutes, small fire 20 minutes, to natural cooling about 1h；
C, be denaturalized: by step b, treated that histotomy PBS is washed 5 minutes/3 times, and 2MHCL is added to act on 30 points at 4 DEG C
Clock；
D, renaturation: by step c, treated that histotomy PBS is washed 5 minutes/3 times, adds 0.01M Tris-HCL at room temperature
Reaction 10 minutes；
E, close: by step d, treated that histotomy PBS is washed 5 minutes/3 times, and lowlenthal serum is closed 2 hours；
F, incubate primary antibody: by step e, treated that histotomy gets rid of extra lowlenthal serum, does not wash, adds BrdU, BrdU/
4 DEG C of DCX antibody overnight incubations；
G, it incubates secondary antibody: by step f treated 37 DEG C of histotomy rewarming 1 hour, adding 37 DEG C of secondary antibody to be incubated for 1 hour；
H, check: by step g, treated that histotomy PBS is washed 5 minutes/3 times, and DAPI redyes karyon 5-8 minutes；
I, mounting: by step h, treated that histotomy PBS washes 5 minutes/3 times, glycerol mounting.
The beneficial effects of the present invention are: the method for the present invention improves conventional immunofluorescence dyeing method, in original
In method the step for " denaturation ", the reaction temperature of 2M HCL is fixed as 4 DEG C, is rebuild using 0.01M Tris-HCL renaturation
Alkaline environment needed for immunofluorescence sufficiently exposes BrdU, and single mark, double marks, three marks dye clear, false positive as the result is shown
It is low.
This modification method has the advantages that (1) simplifies operating process, saves experimental period.Denaturation temperature is fixed as 4
DEG C, renaturation is used uniformly 0.01M Tris-HCL, and controllability is strong, reduces influence factor；(2) dyeing effect is good, and repeatability is high,
Success rate is up to 95% or more；(3) single mark, double marks, three mark dyeing can be carried out, effect is good.Therefore, this modification method has good
Good application prospect and promotional value has substantive distinguishing features outstanding and significant progress.
Detailed description of the invention
To make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with attached drawing to the method for the present invention
It is described in further detail, in which:
Fig. 1 is the BrdU using different denaturation temperature+/DAPI+Immunofluorescence.
Fig. 2 is the BrdU using different denaturation temperature+/DCX+/DAPI+Immunofluorescence.
Specific embodiment
Hereinafter reference will be made to the drawings, and the preferred embodiment of the method for the present invention is described in detail.
The present embodiment carries out SD rat cerebral tissue frozen section using the immunofluorescence dyeing method that the present invention improves
BrdU, DCX, DAPI dyeing, and be compared with conventional method.
Experimental animal: it SD rat 5, is provided by Medical University Of Chongqing's Experimental Animal Center.
Experiment reagent: rabbit-anti mouse BrdU monoclonal antibody and rabbit-anti mouse DCX monoclonal antibody are Abcam Products,
BrdU is Suo Laibao Products, and DAPI is green skies Products, secondary antibody goat antirabbit Alexa Fluor 488488 and mountain
Sheep anti-Mouse Alexa Fluor 594 is biotech firm, Zhong Shan Golden Bridge, and lowlenthal serum confining liquid is biotech firm, Zhong Shan Golden Bridge,
PBS powder is purchased from Wuhan doctor's moral biotech firm.
Experimental facilities: Al+R fluorescence co-focusing (Nikon company, Japan), LIBROR AEG-220 electronic balance
(SHIMADZU company, Japan), inverted microscope (Olympus company, Japan), -80 DEG C of ultra low temperature freezers (Thermo company,
The U.S.)
Experimental method: the following steps are included:
1) intraluminal middle cerebral artery occlusion in rats occlusion/re-perfusion model foundation: import fish is selected in the preparation of rat MCAO/R model
Line, does density bullet at diameter 0.23-0.26mm, long 5.0cm, 1.6-1.8cm, spare after ethanol disinfection.Anesthesia: intraperitoneal injection
After 10% chloraldurate 0.3ml/100g anesthesia.Neck median incision after disinfection separates artery inside and outside the neck summation neck of right side, ligation
Arteria carotis communis proximal part and external carotid artery distal end place artery clamp in internal carotid distal end, and arteria carotis communis crotch is cut off, to
Internal carotid is inserted into fishing line 1.6-1.8cm, ligatures arteria carotis communis and fixed fishing line, stays 10mm the end of a thread, skin suture outside.Ischemic
After 60min, line bolt is exited to progress Reperfu- sion, postoperative maintenance rat temperature, free diet at common carotid artery occlusion.Sham-operation
Group only separates artery inside and outside right carotid and neck, but not plug wire.
2) intraperitoneal injection is after modeling success 30min with 100mg/kg BrdU, once a day, continuous injection 7 days, after modeling
It anaesthetized within 14 days, open chest, aortic cannulation, perfusion takes brain, and 4% paraformaldehyde is fixed for 24 hours, after saccharose gradient dehydration is sunk to the bottom, system
At the brain tissue frozen section of 10um thickness.
3) steps are as follows for fluorescence:
A, rupture of membranes: taking histotomy to dry in the air at room temperature 15 minutes, and then plus 0.4%triton reacts 20 minutes at room temperature.
B, repair: by step a, treated that histotomy PBS is washed 5 minutes/3 times, with 0.01mmlol/L citrate
Progress microwave thermal reparation, high fire 5 minutes, small fire 20 minutes, to natural cooling about 1h.
C, be denaturalized: by step b, treated that histotomy PBS is washed 5 minutes/3 times, and 2M HCL is added to act on 30 at 4 DEG C
Minute.
D, renaturation: by step c, treated that histotomy PBS is washed 5 minutes/3 times, adds 0.01M Tris-HCL at room temperature
Reaction 10 minutes.
E, close: by step d, treated that histotomy PBS is washed 5 minutes/3 times, and lowlenthal serum is closed 2 hours.
F, incubate primary antibody: by step e, treated that histotomy gets rid of extra lowlenthal serum, does not wash, adds BrdU, BrdU/
4 DEG C of DCX monoclonal antibody overnight incubations.
G, it incubates secondary antibody: by step f treated 37 DEG C of histotomy rewarming 1 hour, adding 37 DEG C of secondary antibody to be incubated for 1 hour.
H, check: by step g, treated that histotomy PBS is washed 5 minutes/3 times, and DAPI redyes karyon 5-8 minutes.
I, mounting: by step h, treated that histotomy PBS is washed 5 minutes/3 times, glycerol mounting, is copolymerized burnt observation, claps
According to.
J, count: every rat randomly selects 2 slices, and every slice chooses 2 visuals field and carries out observation statistics.
Experimental result:
The immunofluorescence dyeing of BrdU is carried out using different denaturation temperature methods, the expression of each group is shown in Fig. 1,2.
37 ° of denaturation: BrdU positive mark location ambiguity is indefinite as the result is shown, and double target marker DCX cannot be just therewith
Often colour developing, DAPI also cannot normally develop the color, smudgy, be in cloud (Figure 1A, B, C；Fig. 2A, B, C, D).
Room temperature denaturation: BrdU positive mark positioning is clear as the result is shown, and double target marker DCX colour developings are relatively fuzzy therewith,
DAPI colour developing is unstable, and fluorescent brightness is partially dark, but compared with 37 ° of degeneration methods good (Fig. 1 D, E, F；Fig. 2 E, F, G, H).
4 ° of denaturation: BrdU positive mark positioning is clear as the result is shown, and double target marker DCX color developing effects are good therewith,
DAPI color developing effect, and fluorescent brightness is moderate, is denaturalized (Fig. 1 G, H, I better than room temperature；Fig. 2 I, J, K, L).
In conclusion different denaturation temperature detection result shows that 4 ° of degeneration methods carry out the immunofluorescence dyeing effect of BrdU most
It is good.
Experiment conclusion:
Either singly mark, double marks or three mark dyeing all have extraordinary dyeing effect to the method for the present invention, and setting ratio is high
It is reproducible up to 95% or more, it is easy to operate and save experimental period.
Claims (1)
- The modification method of 1.BrdU labelled immune fluorescence detection cell Proliferation, the specific steps of which are as follows:A, rupture of membranes: taking histotomy to dry in the air at room temperature 15 minutes, and then plus 0.4%triton reacts 20 minutes at room temperature；B, repair: by step a, treated that histotomy PBS is washed 5 minutes/3 times, is carried out with 0.01mmol/L citrate micro- Wave hot repair is multiple, and high fire 5 minutes, small fire 20 minutes, to natural cooling 1h；C, be denaturalized: by step b, treated that histotomy PBS is washed 5 minutes/3 times, and 2M HCL is added to act on 30 minutes at 4 DEG C；D, renaturation: by step c, treated that histotomy PBS is washed 5 minutes/3 times, and 0.01M Tris-HCL is added to react at room temperature 10 minutes；E, close: by step d, treated that histotomy PBS is washed 5 minutes/3 times, and lowlenthal serum is closed 2 hours；F, incubate primary antibody: by step e, treated that histotomy gets rid of extra lowlenthal serum, does not wash, adds BrdU, DCX/BrdU mono- 4 DEG C of antibody overnight incubations；G, it incubates secondary antibody: by step f treated 37 DEG C of histotomy rewarming 1 hour, adding 37 DEG C of secondary antibody to be incubated for 1 hour；H, check: by step g, treated that histotomy PBS is washed 5 minutes/3 times, and DAPI redyes karyon 5-8 minutes；I, mounting: by step h, treated that histotomy PBS washes 5 minutes/3 times, glycerol mounting；It is characterized by: denaturation temperature is fixed as 4 DEG C, renaturation is used uniformly 0.01M Tris-HCL, carry out single mark, double marks and Three mark dyeing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201610646905.5A CN107703292B (en) | 2016-08-09 | 2016-08-09 | The modification method of BrdU labelled immune fluorescence detection cell Proliferation |
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