This experiment labeled with peroxidase assay apoptosis. The method can be used for formalin-fixed paraffin-embedded tissue sections, frozen sections, and apoptosis assays that are cultured or isolated from tissue. Experimental principle Deoxyribonucleotide derivative digoxigenin-11-dUTP can be incorporated into the 3-OH end of double-stranded or single-stranded DNA of apoptotic cells under the action of TdT enzyme, which is much different from dATP. It is a polymer and can bind to an anti-digoxigenin antibody linked to a reporter enzyme (peroxidase or alkaline phosphatase). In the presence of a suitable substrate, peroxidase produces a strong color response, specifically and accurately localizes the cells that are undergoing apoptosis, and thus can be observed under ordinary light microscopy. Foxglove plants are the only source of digoxin. There is almost no ligand that binds to the anti-digoxigenin in all animal tissues, and thus the non-specific reaction is very low. The cross-reactivity of the anti-digoxigenin-specific antibody with the vertebrate steroid hormone is less than 1%. If the Fc portion of the antibody is removed by protease hydrolysis, the non-specific adsorption of the Fc receptor can be completely ruled out. Primary reagent 1. Phosphate buffer PBS (pH 7.4): sodium phosphate 50 mM, NaCl 200 mM. 2. Proteinase K (200 μg/ml, pH 7.4): proteinase K 0.02 g; PBS 100 ml. 3. PBS buffer (pH 7.4) containing 2% H2O2: H2O22.0 ml; PBS buffer 98.0 ml. 4. TdT enzyme buffer (fresh formulation): 3.63 g of Trzz base was adjusted to pH 7.2 with 0.1 N HCl, and made up to 1000 ml with ddH 2 O; then 29.96 g of sodium dimethyl arsenate and 0.238 g of cobalt chloride were added. 5. TdT enzyme reaction solution: 32 μl of TdT enzyme; 76 μl of TdT enzyme buffer, mix and place on ice for use. 6. Wash and stop the reaction buffer: sodium chloride 17.4 g; sodium citrate 8.82 g; ddH2O 1000 ml. 7. 0.05% diaminobiphenyl (DAB) solution: DAB 5 mg; PBS 10 ml, pH 7.4, and filtered before use, hydrogen peroxide was added to 0.02%. 8. 0.5% methyl green (pH 4.0): methyl green 0.5 g; 0.1 M sodium acetate 100 ml. 9. 100% butanol, 100%, 95%, 90%, 80% and 70% ethanol, xylene, 10% neutral formaldehyde solution, acetic acid, pine perfume, etc. 10. Peroxidase-labeled anti-digoxigenin antibody (ONCOR). Experimental procedure 1. Specimen pretreatment: 1) Paraffin-embedded tissue section pretreatment: Tissue sections were placed in a dyeing tank and washed twice with xylene for 5 min each time. Wash twice with absolute ethanol for 3 min each time. Wash once with 95% and 75% ethanol for 3 min each time. The proteinase K solution (20 ug/ml) was added for 5 min with PBS, and hydrolyzed at room temperature for 15 min to remove tissue proteins. Wash 4 times with distilled water for 2 min each time, then proceed as follows. 2) Pretreatment of frozen tissue sections: The frozen tissue sections were placed in 10% neutral formaldehyde and fixed at room temperature for 10 min to remove excess liquid. Wash twice with PBS for 5 min each time. Place the solution in ethanol: acetic acid (2:1) at -20 ° C for 5 min to remove excess liquid. Wash twice with PBS for 5 min each, then follow step 2 below. 3) Pretreatment of cultured or isolated cells: Approximately 5 x 107 cells/ml of cells were fixed in 4% neutral formaldehyde for 10 min at room temperature. 50-100 μl of the cell suspension was added dropwise to the slide and allowed to dry. Wash twice with PBS for 5 min each, then follow step 2 below. 2. Add 2% hydrogen peroxide in PBS to the color cylinder and react at room temperature for 5 min. Wash twice with PBS for 5 min each time. 3. Using a filter paper, carefully remove excess liquid from the tissue on the slide. Immediately add 2 drops of TdT enzyme buffer to the section and let it stand for 1 to 5 minutes at room temperature. 4. Carefully aspirate excess liquid around the section with filter paper, immediately add 54 μl of TdT enzyme reaction solution to the section, and allow to react at 37 ° C for 1 hr in a humid chamber (note: negative staining control, add reaction solution without TdT enzyme). 5. Place the sections in the dyeing tank, add the washing and termination reaction buffer that has been preheated to 37 ° C, incubate at 37 ° C for 30 min, gently lift and slide the slides every 10 min to make the liquid slightly agitate. 6. Tissue sections were washed 3 times with PBS, and after 5 min each, two drops of peroxidase-labeled anti-digoxigenin antibody were directly added to the sections, and reacted in a wet box at room temperature for 30 min. 7. Wash 4 times with PBS for 5 min each time. 8. Add freshly prepared 0.05% DAB solution directly onto the tissue section and develop color for 3-6 minutes at room temperature. 9. Wash 4 times with distilled water, 1 min for the first 3 times and 5 min for the last one. 10. Counterstain with methyl green for 10 min at room temperature. Wash 3 times with distilled water, lift the slides 10 times for the first two times, and let stand for 30 s for the last time. It was washed three times with 100% n-butanol in the same manner. 11. Dehydrate with xylene three times for 2 min each time, seal and dry, observe and record the results under an optical microscope. Precautions Be sure to set up positive and negative cell controls. The positive control sections can be partially degraded using DNase I, and the positive cell controls can be treated with dexamethasone (1 μM) for 3-4 hr of large, mouse thymocytes or human peripheral blood lymphocytes. The negative control did not add TdT enzyme, and the remaining steps were the same as the experimental group.Liver protection
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