CN107703292A - The modification method of BrdU labelled immune fluoroscopic examinations cell propagation - Google Patents

Abstract

The invention discloses an improved method for detecting BrdU-labeled proliferating cells by immunofluorescence. The denaturation temperature was fixed at 4°C, renaturation was uniformly treated with 0.01M Tris-HCL, and then single-, double-, and triple-labeled staining was performed, and compared with conventional methods. The result shows that the method of the invention has very good dyeing effect, high success rate, good repeatability, simple operation and saves experiment time.

Description

An Improved Method for Detection of Cell Proliferation by BrdU-labeled Immunofluorescence

technical field

The invention relates to the field of biology, in particular to an improved method for detecting BrdU-labeled proliferating cells by immunofluorescence.

Background technique

5-bromodeoxyuridine (BrdU) is a synthetic analogue of thymine. During the DNA synthesis phase (S phase), BrdU can replace thymine and infiltrate into the replicating DNA molecule, while there is no endogenous tissue cell Sexual BrdU is present. BrdU was added in cell culture or in vivo injection, followed by immunofluorescence staining, BrdU positive cells were proliferating cells. At the same time, combined with other cell markers and double staining, it can qualitatively and quantitatively detect the types of proliferating cells, proliferation speed and differentiation, migration and survival of proliferating cells, which is of great significance to the study of cell dynamics. BrdU-labeled immunofluorescence detection of cell proliferation is widely used in various cell proliferation studies.

At present, there are various staining methods for BrdU-labeled immunofluorescence detection of cell proliferation, and there is no uniform procedure. Below are the steps for two commonly used staining methods.

The steps of the first dyeing method are as follows:

a. Membrane rupture: take tissue slices, add 0.4% triton and react at room temperature for 20 minutes;

b. Restoration: The tissue slices treated in step a were washed with PBS for 5 minutes/3 times, and microwave thermal repair was performed with 0.01mmlol/L citrate, with high heat for 5 minutes, low heat for 20 minutes, and natural cooling for about 1 hour;

c. Denaturation: Wash the tissue sections treated in step b with PBS for 5 minutes/3 times, add 2M HCL and act at 37°C for 30 minutes;

d. Refolding: wash the tissue sections treated in step c with PBS for 5 minutes/3 times, then add 0.01M Tris-HCL to react at room temperature for 10 minutes;

e. Sealing: wash the tissue sections treated in step d with PBS for 5 minutes/3 times, and seal with goat serum for 2 hours;

f. Incubate the primary antibody: remove excess goat serum from the tissue slices treated in step e, without washing, add BrdU and its double-labeled antibody and incubate overnight at 4°C;

g. Incubate with secondary antibody: rewarm the tissue section at 37°C for 1 hour after the treatment in step f, add secondary antibody and incubate at 37°C for 1 hour;

h. Recheck: Wash the tissue section with PBS for 5 minutes/3 times after the treatment in step g, and counterstain the nucleus with DAPI for 5-8 minutes;

i. Mounting: wash the tissue sections treated in step h with PBS for 5 minutes/3 times, and seal with glycerol.

The steps of the second dyeing method are as follows:

a. Denaturation: take tissue slices, add 1M HCL and incubate on ice for 10 minutes, then add 2M HCL and incubate at room temperature for 10 minutes, then incubate at 37°C for 20 minutes;

b. Refolding: add 0.1M boric acid buffer solution to the tissue section treated in step a and neutralize for 10 minutes at room temperature;

c. Membrane rupture: take tissue slices, add 0.4% triton and react at room temperature for 20 minutes;

d. Restoration: The tissue slices treated in step a were washed with PBS for 5 minutes/3 times, and microwave thermal repair was performed with 0.01mmlol/L citrate, with high heat for 5 minutes, low heat for 20 minutes, and natural cooling for about 1 hour;

e. Sealing: wash the tissue sections treated in step d with PBS for 5 minutes/3 times, and seal with goat serum for 2 hours;

f. Incubate the primary antibody: remove excess goat serum from the tissue slices treated in step e, without washing, add BrdU and its double-labeled antibody and incubate overnight at 4°C;

g. Incubate with secondary antibody: rewarm the tissue section at 37°C for 1 hour after the treatment in step f, add secondary antibody and incubate at 37°C for 1 hour;

h. Recheck: Wash the tissue section with PBS for 5 minutes/3 times after the treatment in step g, and counterstain the nucleus with DAPI for 5-8 minutes;

i. Mounting: wash the tissue sections treated in step h with PBS for 5 minutes/3 times, and seal with glycerol. The above two methods are currently commonly used immunofluorescence staining methods, which have the following disadvantages:

1. The denaturation temperature is not fixed. The first method directly adopts 37° denaturation, and the location of the BrdU positive marker is blurred and unclear. The second marker cannot be normally colored during double-labeled staining, and the nucleus cannot be clearly displayed by DAPI or PI staining. The second method uses 10 minutes on ice → 10 minutes at room temperature → 20 minutes at 37°C to treat cells or tissues for denaturation. The operation process is complicated. . Our previous experiments have also verified that the results at room temperature in winter are better, while the results at room temperature in summer show that DAPI is difficult to stain the nucleus.

2. Different refolding methods. The first method uses 0.01M Tris-HCL renaturation, and the second method uses 0.1M boric acid renaturation. There are also ways to use non-reversion. Our experiments have verified that the renaturation effect is best with 0.01M Tris-HCL.

3. The steps are cumbersome and inconsistent.

4. The above two conventional methods make BrdU single-labeling staining effect extremely poor, and double-labeling and nuclear staining cannot be implemented, which seriously affects the experimental results. Therefore, this method needs to be improved.

Invention content:

In view of this, the purpose of the present invention is to provide an improved BrdU-labeled immunofluorescence staining method for detecting cell proliferation, which can perform single-label, double-label, and triple-label staining. The staining effect is clear and repeatable, and the method is simple and economical. Strong controllability, few unfavorable factors.

The method steps of the improved BrdU-labeled immunofluorescent staining of the present invention to detect cell proliferation are as follows:

a. Membrane rupture: take tissue slices and let it dry for 15 minutes at room temperature, then add 0.4% triton and react for 20 minutes at room temperature;

b. Restoration: The tissue slices treated in step a were washed with PBS for 5 minutes/3 times, and microwave thermal repair was performed with 0.01mmlol/L citrate, with high heat for 5 minutes, low heat for 20 minutes, and natural cooling for about 1 hour;

c. Denaturation: wash the tissue sections treated in step b with PBS for 5 minutes/3 times, add 2M HCL and act at 4°C for 30 minutes;

d. Refolding: wash the tissue slices treated in step c with PBS for 5 minutes/3 times, add 0.01M Tris-HCL and react at room temperature for 10 minutes;

e. Sealing: wash the tissue sections treated in step d with PBS for 5 minutes/3 times, and seal with goat serum for 2 hours;

f. Incubate with primary antibody: remove excess goat serum from tissue slices treated in step e, without washing, add BrdU and BrdU/DCX antibodies and incubate overnight at 4°C;

g. Incubate with secondary antibody: rewarm the tissue section at 37°C for 1 hour after the treatment in step f, add secondary antibody and incubate at 37°C for 1 hour;

h. Recheck: Wash the tissue section with PBS for 5 minutes/3 times after the treatment in step g, and counterstain the nucleus with DAPI for 5-8 minutes;

i. Mounting: wash the tissue sections treated in step h with PBS for 5 minutes/3 times, and seal with glycerol.

The beneficial effect of the present invention is that: the method of the present invention improves the conventional immunofluorescent staining method. In the step of "denaturation" in the original method, the reaction temperature of 2M HCL is fixed at 4°C, and then recombined with 0.01M Tris-HCL The basic environment required for permanent reconstruction of immunofluorescence, fully exposed to BrdU, the results showed that the single-label, double-label, and triple-label staining were clear and the false positives were low.

This improved method has the following advantages: (1) The operation process is simplified and the experiment time is saved. The denaturation temperature is fixed at 4°C, and 0.01M Tris-HCL is uniformly used for refolding, which is highly controllable and reduces the influencing factors; (2) the dyeing effect is good, the repeatability is high, and the success rate is as high as 95%; (3) it can Single-, double-, and triple-standard staining was performed with good results. Therefore, this improved method has good application prospects and promotion value, and has outstanding substantive features and remarkable progress.

Description of drawings

In order to make the purpose of the present invention, technical solutions and advantages clearer, the method of the present invention will be further described in detail below in conjunction with the accompanying drawings, wherein:

Figure 1 is the immunofluorescence diagram of BrdU ⁺ /DAPI ⁺ using different denaturation temperatures.

Figure 2 is the immunofluorescence diagram of BrdU ⁺ /DCX ⁺ /DAPI ⁺ at different denaturation temperatures.

Detailed ways

Preferred embodiments of the method of the present invention will be described in detail below with reference to the accompanying drawings.

In this example, the improved immunofluorescent staining method of the present invention was used to stain SD rat brain tissue frozen sections with BrdU, DCX, and DAPI, and compared with conventional methods.

Experimental animals: 5 SD rats, provided by the Experimental Animal Center of Chongqing Medical University.

Experimental reagents: Rabbit anti-mouse BrdU monoclonal antibody and rabbit anti-mouse DCX monoclonal antibody are products of Abcam, BrdU is a product of Suleibao Company, DAPI is a product of Beyond Company, secondary antibodies goat anti-rabbit Alexa Fluor 488488 and goat anti-small Mouse Alexa Fluor 594 was purchased from Zhongshan Jinqiao Biological Company, goat serum blocking solution was purchased from Zhongshan Jinqiao Biological Company, and PBS powder was purchased from Wuhan Boster Biological Company.

Experimental equipment: Al+R fluorescence confocal (Nikon Company, Japan), LIBROR AEG-220 electronic balance (SHIMADZU Company, Japan), inverted microscope (Olympus Company, Japan), -80 ℃ ultra-low temperature refrigerator (Thermo Company, USA)

Experimental method: including the following steps:

1) Establishment of rat middle cerebral artery occlusion/reperfusion model: Rat MCAO/R model was prepared by using imported fishing line with a diameter of 0.23-0.26mm and a length of 5.0cm. Black marks were made at 1.6-1.8cm, and the reserve was sterilized with ethanol. use. Anesthesia: intraperitoneal injection of 10% chloral hydrate 0.3ml/100g after anesthesia. Disinfect the median incision in the posterior neck, separate the right common carotid artery and internal and external carotid artery, ligate the proximal end of the common carotid artery and the distal end of the external carotid artery, place an arterial clip at the distal end of the internal carotid artery, and cut open the bifurcation of the common carotid artery. Insert the fishing line 1.6-1.8cm into the internal carotid artery, ligate the common carotid artery and fix the fishing line, leave 10mm of thread outside, and suture the skin. After 60 minutes of ischemia, the thread plug was withdrawn to the ligated part of the common carotid artery for reperfusion. After the operation, the body temperature of the rats was maintained and they were free to eat and drink. In the sham operation group, only the right common carotid artery and the internal and external carotid arteries were separated, but the line was not inserted.

2) 30 minutes after successful modeling, 100 mg/kg BrdU was injected intraperitoneally, once a day, for 7 consecutive days. After 14 days after modeling, anesthesia, thoracotomy, aortic cannulation, brain perfusion, and 4% paraformaldehyde fixation for 24 hours , after sucrose gradient dehydration and sinking to the bottom, 10um thick frozen sections of brain tissue were made.

3) The fluorescence steps are as follows:

a. Membrane rupture: Take tissue slices and let them dry at room temperature for 15 minutes, then add 0.4% triton and react at room temperature for 20 minutes.

b. Restoration: The tissue slices treated in step a were washed with PBS for 5 minutes/3 times, and microwave heat repair was performed with 0.01mmlol/L citrate, with high heat for 5 minutes, low heat for 20 minutes, and natural cooling for about 1 hour.

c. Denaturation: Wash the tissue sections treated in step b with PBS for 5 minutes/3 times, add 2M HCL and act at 4°C for 30 minutes.

d. Refolding: Wash the tissue sections treated in step c with PBS for 5 minutes/3 times, add 0.01M Tris-HCL and react at room temperature for 10 minutes.

e. Blocking: wash the tissue sections treated in step d with PBS for 5 minutes/3 times, and block with goat serum for 2 hours.

f. Incubate with primary antibody: Remove excess goat serum from tissue sections treated in step e, without washing, add BrdU and BrdU/DCX monoclonal antibody and incubate overnight at 4°C.

g. Secondary antibody incubation: after the treatment in step f, the tissue sections were rewarmed at 37°C for 1 hour, and then added with secondary antibody and incubated at 37°C for 1 hour.

h. Recheck: wash the tissue section with PBS for 5 minutes/3 times after the treatment in step g, and counterstain the nucleus with DAPI for 5-8 minutes.

i. Mounting: wash the tissue sections treated in step h with PBS for 5 minutes/3 times, seal with glycerol, observe by confocal, and take pictures.

j. Statistics: 2 slices were randomly selected for each rat, and 2 fields of view were selected for each slice for observation and statistics.

Experimental results:

Immunofluorescence staining of BrdU was carried out using different denaturation temperature methods, and the expression of each group is shown in Figures 1 and 2.

37° degeneration: The results showed that the location of the BrdU positive marker was vague and unclear, and the double-labeled marker DCX could not develop color normally, and neither could DAPI, which was blurred and cloudy (Fig. 1A, B, C; Fig. 2A , B, C, D).

Denaturation at room temperature: The results showed that the positive marker of BrdU was clearly positioned, the color of the double-labeled marker DCX was blurred, the color of DAPI was unstable, and the fluorescence brightness was dark, but it was better than the 37° denaturation method (Fig. 1D, E, F; Fig. 2E, F, G, H).

4° denaturation: the results showed that the positive marker of BrdU was clearly positioned, and the double-labeled marker DCX and DAPI had good color development effects, and the fluorescence brightness was moderate, which was better than normal temperature denaturation (Fig. 1G, H, I; Fig. 2I , J, K, L).

In summary, the results of different denaturation temperatures showed that the 4° denaturation method was the best for BrdU immunofluorescence staining.

Experimental results:

The method of the invention has very good dyeing effect no matter it is single-standard, double-standard or triple-standard dyeing, the yield rate is as high as over 95%, the repeatability is good, the operation is simple and the experiment time is saved.

Claims (6)

1. An improved method for BrdU-labeled immunofluorescence detection of cell proliferation, characterized in that the denaturation temperature is fixed at 4°C, 0.01M Tris-HCL is uniformly used for refolding, and single-, double-, and triple-labeled staining is performed. 2. the feature of dyeing method according to claim 1, its concrete steps are as follows: a. Membrane rupture: Take tissue slices and let them dry at room temperature for 15 minutes, then add 0.4% triton and react at room temperature for 20 minutes. b. Restoration: The tissue slices treated in step a were washed with PBS for 5 minutes/3 times, and microwave heat repair was performed with 0.01mmlol/L citrate, with high heat for 5 minutes, low heat for 20 minutes, and natural cooling for about 1 hour. c. Denaturation: Wash the tissue sections treated in step b with PBS for 5 minutes/3 times, add 2M HCL and act at 4°C for 30 minutes. d. Refolding: Wash the tissue sections treated in step c with PBS for 5 minutes/3 times, add 0.01M Tris-HCL and react at room temperature for 10 minutes. e. Blocking: wash the tissue sections treated in step d with PBS for 5 minutes/3 times, and block with goat serum for 2 hours. f. Incubate with primary antibody: Remove excess goat serum from tissue sections treated in step e, without washing, add BrdU, DCX/BrdU monoantibody and incubate overnight at 4°C. g. Secondary antibody incubation: after the treatment in step f, the tissue sections were rewarmed at 37°C for 1 hour, and then added with secondary antibody and incubated at 37°C for 1 hour. h. Recheck: wash the tissue section with PBS for 5 minutes/3 times after the treatment in step g, and counterstain the nucleus with DAPI for 5-8 minutes. i. Mounting: wash the tissue sections treated in step h with PBS for 5 minutes/3 times, and seal with glycerol. 3. according to claim 1 and the improved method of BrdU labeling immunofluorescence detection cell proliferation described in claim 1 and 2, it is characterized in that: intraperitoneal injection of 100mg/kg BrdU after rat cerebral ischemia/reperfusion (MCAO/R) injury 30min, every day One time, continuous injection for 7 days, and 14 days after modeling, the brain was perfused. 4. according to claim 1 and the improved method of BrdU labeling immunofluorescence detection cell proliferation described in claim 1 and 2, it is characterized in that: 5-bromo-2-deoxyuridine (5-Bromo-2-deoxyUridine, BrdU) is a A thymidine analog that is integrated into the nucleus during DNA synthesis in the S phase of the cell cycle and can be detected by immunohistochemistry, can be used to label cells undergoing DNA synthesis during BrdU exposure, so BrdU is the response Very reliable marker of cell proliferation. 5. The improved method for detecting cell proliferation by BrdU-labeled immunofluorescence according to claims 1 and 2, characterized in that: the denaturation temperature of 2M hydrochloric acid is 4°C. 6. The improved method of BrdU-labeled immunofluorescence detection of cell proliferation according to claims 1 and 2, characterized in that: 2M hydrochloric acid makes DNA double-strand denaturation unravel, Tris-HCL rebuilds the required alkaline environment for immunohistochemistry, Fully expose BrdU.