Human Bone Alkaline Phosphatase II. Precautions 1. This reagent is an in vitro test reagent. It should be used within the validity period. The reagent should be regarded as an infectious agent. Reagents with different batch numbers should not be mixed. The reagents in the box should be taken out before use. Leave at room temperature for at least 30 minutes. After the concentrated washing liquid crystallizes, incubate at 37 Â° C for 15 minutes. After the concentrated sample diluent crystallizes, please incubate at 37 â„ƒ for 15 minutes. If the experiment is performed within 24 hours, the specimen can be stored at 2 ï½ž 8 â„ƒ. No need for timely experiment, the specimen should be stored at -20 â„ƒ to avoid repeated freezing and thawing. After repeatedly cleaning the microwell plate, and buckle the residual liquid in the microwell, otherwise it will reduce the accuracy and cause the false image of absorbance deviation. After the sample is added, the microwell reaction strip should be shaken slightly to mix the liquid in the well. The kit is stored at 2 ï½ž 8 â„ƒ, please do not freeze it, please refer to the label in the box for the expiration date. Human bone alkaline phosphatase III. Preparation before experiment 1. Before use, the reagents in the box should be taken out and left at room temperature for at least 30 minutes. 2. 2. Prepare various experimental instruments and materials, such as micropipettes, pipette tips, medical distilled water, etc. 3. 3. Concentrated lotion and medical distilled water are diluted 1:19 times to become the applied lotion 4. 4. Dilute the concentrated sample diluent and medical distilled water 1: 4 times into the applied sample diluent 5. Dilute the sample with the application sample diluent, dilute the sample according to the volume ratio of 1: 100, for example, 10Î¼l of the sample is added to the 1ml application sample diluent, and mix well to use. 4) Operation steps Remove the enzyme labeling plate, set up blank wells, add 100Î¼l of standard products to the blank microwells according to the order (the blank wells are regarded as No. 0 standard, and use medical distilled water to replace) 2. Mark the sample number separately and add 100Î¼l of diluted sample In blank microwells (different samples use different tips). 3. Incubate the microtiter plate at 37 Â° C for 30 minutes; 4. Remove the microtiter plate and shake off the liquid. After each well is filled with application washing liquid, immediately shake off the liquid; 5. After each well is filled with the application washing solution, shake the enzyme plate slightly for 30 seconds, then shake off the application washing solution in the wells, and pat the enzyme plate on the absorbent paper to dry. 6. Repeat the fifth step 5 times, pat the enzyme plate dry on absorbent paper. 7. Add 100 Î¼l of enzyme-labeled coupling solution to the standard well and sample well. 8. Incubate the 96-well plate at 37 Â° C for 30 minutes. 9. Take out the enzyme labeling plate and shake off the liquid. After all the application washing liquid is filled in each well, immediately shake off the liquid. 10. After refilling each well with the washing application solution, shake the enzyme plate slightly at room temperature for 30 seconds, then shake off the application washing solution in the wells, and pat the enzyme plate on the absorbent paper to dry. 11. Repeat the fifth step 5 times, pat the enzyme plate on the absorbent paper to dry. 12. Add 50 Î¼l of substrate A to each well immediately after adding 50 Î¼l of substrate B. Gently shake to mix. (Liquid A and B are added with different tips) 13. Incubate the microtiter plate at 37 Â° C in the dark for 15 minutes. 14. Add 50 Î¼l of stop solution to each microwell and gently shake to mix. 15. Measure OD at 450nm on a microplate reader; after color development, measure within 15 minutes. 16. Calculate the sample content of human bone alkaline phosphatase according to the prepared standard curve. V. Judgment of results 1. Instrument value: read the OD value of each well on a microplate reader with a wavelength of 450 nm; 2. Range of detection values: 0 â€“ 1200U / L 3. Sensitivity: 1.0 U / LMCM7 (minichromosome maintenance protein 7) Microchromosome maintenance defect protein 7 antibody 0.2mlM-CSF (Macrophage Colony Stimulating Factors) Macrophage clone stimulating factor antibody 0.1mlM-CSF (Macrophage Colony Stimulating Factors) ) Macrophage clone stimulating factor antibody 0.2mlMcl-1 (myeloid cell leukemia 1) Myeloid leukemia-1 antibody 0.2mlMDM2 (urine double minute 2) double microsome 2 oncogene antibody 0.1mlMDM2 (urine double minute 2) double micro Body 2 oncogene antibody 0.2ml Streptavidin / APC fluorescein APC labeled streptavidin 0.1ml
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