In situ terminal transferase labeling of apoptotic cells (TUNEL method)
The final step of the multi-step mechanism of apoptosis is: the activation of endogenous endonucleases in the cell leads to the breakage of the double strand of nuclear chromatin DNA. The 3 hydroxyl groups exposed by a large number of DNA fragments are combined with biotin or digoxin-labeled nucleotides under the action of terminal deoxynucleotidyltransferase (TdT) or DNA polymerase, and finally with the aid of antibodies to albumin or digoxin The combined fluorescein or HRP allows apoptotic cells to be specifically labeled and displayed.
TUNEL apoptosis kit is provided by Roche
Reagent 1: Enzyme Solution
Reagent 2: Labeling Solution (LableSolution)
Reagent 3: Converter-POD (Converter-POD)
Enzyme-labeled anti-fluorescein antibody (ready-to-use)
Other reagents required for the test:
Non-paraffin section:
Rinse buffer: phosphate buffered saline (PBS)
Blocking solution: 0.3% H2O2 methanol solution
Fixing solution: 4% paraformaldehyde (solvent pH7.4 freshly prepared PBS solution)
Permeate: 0.1% Triton ç”” -100 (dissolved in freshly prepared 0.1% sodium citrate solution)
Paraffin section:
Xylene and ethanol (100%, 95%, 90%, 80%, 70% diluted with distilled water)
Rinse buffer: phosphate buffered saline (PBS)
· Protease K working solution 10 ~ 20mg / ml dissolved in 10mMTris / HCl (pH7.4 ~ 7.8)
Choose according to your needs:
Permeate: 0.1% TritonX-100 (dissolved in freshly prepared 0.1% sodium citrate solution)
· Gastric enzyme solution (0.25% -0.5% soluble in HCl, pH 2) or pancreatin
· 0.1M citrate buffer, pH6, microwave repair
Materials: Microwave oven, microwave output power 850W-2000W 2. Use biopsy and laboratory animal specimens fixed with neutral methanol, conventional dehydration, xylene transparent, paraffin embedded.
Steps
Antigen microwave repair (for reference)
1. After formalin fixation, tissues will produce a wide range of protein cross-links, so paraffin tissues should be pre-treated for apoptosis detection. According to the long-term research of our company ’s scientific research personnel, the role of proteinase K during TUNEL staining may cause the release of endogenous endonuclease, causing a false positive reaction. At the same time, excessive treatment with proteinase K may destroy the structure of the tissue. Substituting the above processing can avoid the above-mentioned drawbacks.
2. Microwave has been used more and more as a pretreatment process for immunohistochemistry and in situ hybridization. Microwave treatment at a certain temperature can repair the antigen damage caused by immobilization and embedding, interrupt the peptide bond of amino acids, and promote The exposure of antigenic determinants restores the spatial structure of the cell membrane, increases the penetration effect, and accelerates the tissue processing and staining process. We perform microwave repair before TUNEL staining, which can achieve the effect of proteinase K and achieve a more ideal effect. The background staining is very light, and the positive particles of apoptotic cells are in sharp contrast with the background staining.
3. The conditions for the action of proteinase K are strict, and the digestibility is often inconvenient to grasp due to the difference in tissue. At the same time, proteinase K is expensive, and microwave has been used as a common instrument for immunohistochemical staining.
(1) Slice routinely dewaxed into water
(2) Place 200ml of 0.01M, pH 6.0 citric acid buffer solution in a beaker, heat to 90 ～ 95 ℃, quickly put into slices, use 680W (80% power), microwave irradiation for 1min, add double distilled water ( 20 ~ 25 ℃) 80ml for rapid cooling, move the slide to PBS (20 ~ 25 ℃). (3) Wash with PBS for 5min × 3 times.
(4) Add 20% normal bovine serum for 30 minutes at room temperature.
(5) Add the TUNEL reaction mixture to the slices and incubate at 37 ° C for 90 min (negative pair photo, TDT is not added to the TUNEL mixture).
(6) Wash with PBS 5min × 3 times.
(7) 3% H2O2 methanol solution was blocked for 10 minutes at room temperature.
(8) Incubate at 37 ° C for 90 min.
(9) Add POD conversion agent and incubate at 37 ° C for 30 min.
(10) Wash with PBS 5min × 3 times.
(11) DAB / H2O2 color development.
(12) Hematoxylin light dyeing, conventional dehydration, transparent, and neutral gum sealing.
Results: Those with brown granules in the nucleus were positive cells, combined with morphological features, apoptotic cells could be established. Negative no brown particles on the photo.
Brief introduction of cell apoptosis detection technology (for reference)
1. Morphological changes of apoptotic cells
Cell surface: pseudopodia, microvilli, etc. disappear
Cell body morphology: cells shrink, deform, and become smaller
Nucleus: chromatin is concentrated, the early chromatin gathers in the crescent shape or crescent shape at the nuclear membrane, the late fragmentation
Organelles: dense but morphologically and structurally complete, the early endoplasmic reticulum temporarily expands the cell membrane: intact, and later wraps organelles or nuclear fragments to form apoptotic bodies
Performance in tissues: often scattered as a single cell
The surrounding tissue reaction occurs: apoptotic cells or bodies are engulfed and degraded by adjacent macrophages and epithelial cells, and no inflammatory reaction occurs
Occurrence conditions: gene-controlled programmed cell death, which mostly occurs in organ atrophy, cell-mediated immune killing mechanism, low-dose toxin effect, and various pathophysiological states
2. Morphological detection methods of apoptosis
(1) Light microscope and fluorescence microscope detection of apoptotic cells
1. Producer
(1) Routine histological section, dewaxing and hydration.
(2) The cultured cells can be prepared with a cell centrifuge. Since the apoptotic cells are easily broken during preparation, low-speed centrifugation (250g, centrifugation for 5min) should be used, and the slides should be treated with 1% BSA in advance to make the cells easy to attach. After drying in the air, quickly fix it with 1% paraformaldehyde at 4 ℃ for 15-30min, then transfer to 80% ethanol and fix for 1-2h, store at -20 ℃ until use.
2. Dyeing method
Various methods can be used for dyeing.
(1) Conventional histological staining methods such as Giemsa staining, HE staining or Mayer hematoxylin staining can be used.
(2) Dye with fluorescent dyes, such as DAPI (4 ′, 6′-diamidino-2-phenylindole), PI (propidiumiodide) and 7-AAD (7-aminoacetinomycinD). The staining concentration is: DAPI1-2μg / ml; PI5 -10μg / ml; 7-AAD10-20μg / ml. Since PI dye also binds to RNA, cells should be digested with RNase first (50 Kunitz unit RNase, 15min at 37 ℃ or 30min at room temperature), Then perform PI staining.
3. Results observation
Typical morphological changes of apoptotic cells: cell size shrinkage and deformation; dense nuclear staining and loss of submorphic structure, visible nuclear chromatin is crescent-shaped, or nuclear fragmentation into nuclear fragments of varying sizes; visible on tissue sections Macrophages or adjacent epithelial cells engulf apoptotic cells or apoptotic bodies.
(2) Electron microscopic observation of apoptotic cells
The conventional methods of electron microscopy are used for fixation, embedding and film production. Observation can be done with transmission electron microscope or scanning electron microscope. Under transmission electron microscopy, the nuclear chromatin of apoptotic cells is concentrated, the nuclear membrane disappears, and the endoplasmic reticulum expands, while the morphology of organelles such as mitochondria remains good, and the organelles surrounded by the membrane structure, that is, apoptotic bodies, can be seen. Under the scanning electron microscope, the cell surface structures such as pseudopods and microvilli disappeared, and the cell membrane showed undulations.
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