2. Observation by transmission electron microscope
3. Fluorescence microscope observation 1) Hematoxylin-eosin (He) staining: the nucleus is condensed and broken, blue-black, and the cytoplasm is reddish (apoptotic cells). The normal nucleus is uniform and light blue or blue, and the necrotic nucleus is extremely light blue or blue and disappears.
2) Giemsa staining, Wright staining, etc. The results showed that the color of normal nucleus was uniform, the staining of apoptotic cells became darker, and the staining of necrotic cells was lighter or not. 1) Cells became smaller and completely atrophied.
2) Apoptotic corpuscles are several round corpuscles around cells. The apoptotic cells became smaller and concentrated in cytoplasm.
The morphological changes of nuclear chromatin during apoptosis can be divided into three stages: in the first stage, the nucleus is corrugated or folded, and some chromatin is concentrated; The chromatin in ⅱ a phase is highly concentrated and marginalized; The nucleus of stage Ⅱ b was broken into pieces, and apoptotic bodies were formed. Commonly used fluorescent dyes: acridine orange, PI, DAPI, Hoechst33258, Hoechst33342, EB, etc.
The binding of Hoechst 33342, Hoechst 33258 and DAPI to DNA is non-embedded, mainly in the A-T base region of DNA. When excited by ultraviolet light, it will emit bright blue fluorescence.
The basic principle of 1)PI double staining method
Hoechst is a kind of reactive dye that specifically binds to DNA, which can enter normal cell membrane and has no great cytotoxicity to cells. The fluorescence intensity of Hoechst 33342 in apoptotic cells is higher than that in normal cells.
DAPI is semi-permeable, and is used for the staining of conventional fixed cells.
Propidium iodide (PI) is a nucleic acid dye, which can't penetrate the intact cell membrane, but in the middle and late stages of apoptosis and death, PI can penetrate the cell membrane and dye the nucleus red. Therefore, if Annexin-V matches PI, early and late apoptotic cells and dead cells can be distinguished. Principle: Deoxynucleotide derivative digoxin [(digoxin ligand)-1 1-dutp] can be incorporated into the 3-OH terminal of double-stranded or single-stranded DNA of apoptotic cells under the action of TdT enzyme, and form a heteropolymer with dATP, which can bind to anti-digoxin antibody linked with reporter enzyme (peroxidase or alkaline phosphatase). In the presence of suitable substrate, peroxidase can produce strong color reaction and locate apoptotic cells specifically and accurately, so it can be observed under ordinary optical microscope.
Digitalis is the only source of digoxin. There are almost no ligands that can bind to digoxin in all animal tissues, so the nonspecific reaction is very low. The cross reaction between digoxin specific antibody and vertebrate steroid hormone is less than 65438 0%. If the Fc part of this antibody is removed by protease hydrolysis, the nonspecific adsorption of Fc receptor in cells can be completely ruled out.
This method can be used for the determination of apoptosis in formalin-fixed paraffin-embedded tissue sections, frozen sections and cultured or isolated tissues. 1, phosphate buffer PBS(pH7.4): 50mM sodium phosphate NaCl 200mM NaCl.
2. Protease K(200μg/ml, pH7.4): 0.02g Protease K; PBS 100 ml
3. PBS buffer containing 2%H2O2 (pH 7.4): H2O2 2.0 ml; PBS buffer 98.0 ml
4.TdT enzyme buffer (fresh preparation): 3.63g Trlzma alkali is adjusted to pH 7.2 with 0. 1N HCl, and the volume is constant to1000 ml; DdH2O was used, and then 29.96 g of sodium dimethyl arsenate and 0.238 g of cobalt chloride were added.
5.TdT enzyme reaction solution: TdT enzyme 32 μ l; 76μl TdT enzyme buffer, mix well and put it on ice for later use.
6. Wash the reaction buffer: sodium chloride17.4g; 8.82g sodium citrate; DdH2O 1000 ml
7.0.05% diaminobiphenyl (DAB) solution: DAB 5mg；; PBS 10ml, pH7.4, filtered before use, and added with hydrogen peroxide to 0.02%.
8.0.5% methyl green (pH 4.0): 0.5g; Methyl green; 0. 1M sodium acetate 100ml.
9. 100% butanol, 100%, 95%, 90%, 80%, 70% ethanol, xylene, 10% neutral formaldehyde solution, acetic acid, rosin water, etc.
10, peroxidase labeled anti-digoxin antibody (ONCOR) 1, sample pretreatment:
(1) Pretreatment of paraffin-embedded tissue sections: Place the tissue sections in a dyeing tank and wash them with xylene twice, each time for 5 minutes. Wash twice with anhydrous ethanol for 3min each time. Wash with 95% and 75% ethanol once each for 3min. Wash with PBS for 5 minutes, add protease K solution (20ug/ml), and hydrolyze at room temperature for 65438±5min minutes to remove tissue protein. Wash with distilled water for 4 times, 2 minutes each time, and then follow the following step 2.
⑵ Pretreatment of frozen tissue sections: put frozen tissue sections in 10% neutral formaldehyde, fix them at room temperature for 10min, and then remove excess liquid. Wash it twice with PBS for 5 minutes each time. It was placed in ethanol: acetic acid (2: 1) solution for 5 minutes and treated at -20℃ for 5 minutes to remove excess liquid. Wash it twice with PBS for 5 minutes each time, and then follow step 2 below.
⑶ Pretreatment of cultured or isolated cells: about 5 × 107 cells /ml fixed in 4% neutral formaldehyde at room temperature 10min. Drop 50 ~ 100μ l cell suspension on the glass slide and dry it. Wash it twice with PBS for 5 minutes each time, and then follow step 2 below.
2. Add PBS containing 2% hydrogen peroxide into the colorimetric tank and react for 5min minutes at room temperature. Wash it twice with PBS for 5 minutes each time.
3. Carefully blot the excess liquid around the tissue on the slide with filter paper, immediately add 2 drops of TdT enzyme buffer to the slice, and let it stand at room temperature of 1 ~ 5min.
4. Carefully blot the excess liquid around the slice with filter paper, immediately add 54 μ l of TdT enzyme reaction solution to the slice, and put it into a wet box for reaction at 37C for 65438±0hr (note: negative staining control, add the reaction solution without TDT enzyme).
5. Put the slice into the dyeing tank, add the washing and reaction termination buffer preheated to 37℃, keep the temperature at 37℃ for 30 minutes, gently lift and put down the glass slide every 65438 00 minutes, and slightly stir the liquid.
6. Tissue sections were washed with PBS for 3 times, and after 5 minutes each time, two drops of anti-digoxin antibody labeled with peroxidase were directly added to the sections and reacted at room temperature for 30 minutes in a wet box.
7. Wash with PBS for 4 times, 5min each time.
8. Directly add the freshly prepared 0.05%DAB solution into the tissue section and develop color at room temperature for 3-6 minutes.
9. Rinse with distilled water for 4 times, with the first 3 times being 65438 0 min and the last time being 65438 0 min.
10, counterstained with methyl green at room temperature 10 min. Rinse with distilled water for three times, pull the glass slide up and down 10 times in the first two times, and finally 1 time for 30s. Wash with 100% n-butanol for three times in the same way.
1 1. Dehydrate with xylene for 3 times, 2 minutes each time. After sealing and drying, the experimental results were observed and recorded under an optical microscope. Ensure that positive and negative cell controls are set up. For positive control, samples partially degraded by DNaseI can be used; for positive cell control, thymocytes of rats and mice or human peripheral blood lymphocytes can be treated with dexamethasone (1μM) for 3-4 hours; for negative control, TdT enzyme is not added, and other steps are the same as those of the experimental group.
After irreparable DNA damage, cells usually experience programmed death or apoptosis. However, this mechanism is ineffective in tumor cells, so it can proliferate at will and refuse to accept the order of "suicide". German scientists have discovered the possible reason-tumor cells will degrade a kind of protein which can induce apoptosis. Inhibiting the degradation of this protein can restore the apoptosis mechanism and enhance the curative effect of radiotherapy and chemotherapy. Related papers were published in Nature Cell Biology.
One of the protein molecules that trigger apoptosis after severe DNA damage is HIPK2. Thomas Hofmann of the German Cancer Research Center and his colleagues found that HIPK2 was continuously produced in healthy cells, but it was labeled as "garbage" by an enzyme called Siah- 1, so it was immediately degraded.
Slightly damaged cells will enter a low-level alert state-inhibit the degradation of HIPK2 for a short time. Once the damage is repaired, the cells will immediately resume the degradation of HIPK2. Only in severely damaged cells (for example, both DNA double strands are destroyed) can the degradation of HIPK2 be permanently inhibited. Results HIPK2 accumulated continuously, causing apoptosis and cell suicide.
Researchers speculate that this may be the reason why radiotherapy and chemotherapy sometimes fail. Both treatments will seriously damage tumor cells and eventually lead to programmed death. Thomas Hofmann said: "If there is drug resistance, it is often because tumor cells' refuse' to execute suicide orders."
The researchers inhibited the Siah- 1 enzyme in the experiment, and found that HIPK2 could accumulate and trigger apoptosis even in slightly damaged cells. Hoffman speculated, "Cancer medicine is likely to take advantage of this discovery. For example, we can use Siah- 1 inhibitors combined with radiotherapy or chemotherapy to pull cells back to the apoptosis mechanism.