In situ cell death detection kit

In situ cell death detection kit

(In situ cell death detection kit-POD method)

1. Principle:

TUNEL (TdT-mediated dUTP nick end labeling) apoptosis detection kit is used to detect the nuclear DNA breakage of tissue cells in the early stage of apoptosis. The principle is that dUTP labeled with fluorescein can be connected to the 3'-OH end of the broken DNA in apoptotic cells under the action of deoxyribonucleotide terminal transferase (TdT Enzyme). The fluorescein antibody of HRP (horse-radish peroxidase) specifically binds, and the latter reacts with the HRP substrate diaminobenzidine (DAB) to produce a strong color reaction (dark brown). Apoptotic cells can be observed under an optical microscope; because normal or proliferating cells have almost no DNA breaks, no 3'-OH is formed, and they can rarely be stained. This kit is suitable for the in situ detection of apoptosis at the single cell level of tissue samples (paraffin embedded, frozen and ultrathin sections) and cell samples (cell smears). It can also be used to evaluate the efficacy of antitumor drugs, as well as to determine the type of cell death and differentiation stage through the two-color method.

2. Equipment and reagents

Equipment: optical microscope and its imaging system, small dyeing cylinder, wet box (plastic lunch box and gauze), plastic cover glass or parafilm, straws, samplers and pipette tips of various specifications, etc .;

Reagents: kit contains TdT 10 ×, fluorescein-labeled dUTP 1 ×, HRP labeled with fluorescein antibody; self-prepared reagents: PBS, double distilled water, xylene, gradient ethanol (100, 95, 90, 80, 70% ), DAB working solution (prepared before use, 5 μl 20 × DAB + 1 μL 30% H2O2 + 94 μl PBS), Proteinase K working solution (10-20 μg / ml in 10 mM Tris / HCl, pH 7.4-8) Or cell permeate (0.1% Triton X-100 in 0.1% sodium citrate, prepared before use), hematoxylin or methyl green, DNase 1 (3000 U / ml– 3 U / ml in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mg / ml BSA), etc.

3. Experimental steps

Operation flow chart: making paraffin section → dewaxing and hydration → cell permeability → adding TUNEL reaction solution → adding converter-POD → color development with substrate DAB reaction → counting with optical microscope and taking pictures

Specific steps:

1. Soak twice with xylene, 5min each time;

2. Dip and wash with gradient ethanol (100, 95, 90, 80, 70%) for 3min each time;

3. Rinse twice with PBS;

4. Treat the tissue with Proteinase K working solution for 15-30 min at 21–37 ° C (temperature, time, and concentration must be explored) or add cell permeation solution for 8 min;

5. Rinse twice with PBS;

6. Prepare the TUNEL reaction mixture. The treatment group is mixed with 50μl TdT + 450μl fluorescein-labeled dUTP solution; while the negative control group is only added with 50μl fluorescein-labeled dUTP solution. ~ 25 ℃ × 10min, the following steps are the same as the treatment group.

7. After the slides have dried, add 50 μl of TUNEL reaction mixture (negative control group only adds 50 μl of fluorescein-labeled dUTP solution) to the specimens, add coverslips or parafilm to react in a dark wet box at 37 ℃ × 1h.

8. Rinse 3 times with PBS;

9. Add 1 drop of PBS to count apoptotic cells under a fluorescent microscope (excitation light wavelength 450 ~ 500nm, detection wavelength 515 ~ 565nm);

10. After the slides have dried, add 50 μl of converter-POD to the specimen, add a cover slip or parafilm to react in a dark wet box at 37 ° C for 30 min.

11. Rinse 3 times with PBS;

12. Add 50 ~ 100μl DAB substrate to the tissue and react at 15 ~ 25 ℃ × 10min;

13. Rinse 3 times with PBS;

14. After taking photos, counterstain with hematoxylin or methyl green, and rinse with tap water immediately after a few seconds. Gradient alcohol dehydration, xylene transparent, neutral gum sealing.

15. Add a drop of PBS or glycerol under the visual field, observe the apoptotic cells (200-500 cells in total) with a light microscope and take a picture. It can be combined with the morphological characteristics of apoptotic cells to make a comprehensive judgment (unstained cells become smaller, the membrane is intact but foaming occurs, apoptotic bodies appear in the late stage, and adherent cells appear shrinking, rounding, and shedding; stained cells appear stained Mass concentration, marginalization, nuclear membrane lysis, chromatin segmentation into massive / apoptotic bodies)

4. Matters needing attention

1. When washing with PBS, wash for 5 min each time.

2. After washing with PBS, in order to carry out various reactions effectively, please try to remove the PBS solution before proceeding to the next reaction.

3. After adding the experimental reaction solution to the sample on the slide, please cover the cover slip or plastic wrap, or in a wet box, so that the reaction solution can be evenly distributed throughout the sample, and can also prevent the reaction solution Drying caused the experiment to fail.

4. Prepare the TUNEL reaction solution immediately before use and store it on ice for a short time. Not suitable for long-term storage, long-term storage will lead to inactivation of enzyme activity.

5. If the color of the 20 × DAB solution becomes darker than purple, it cannot be used and needs to be reconstituted.

6. After staining with Methyl Green staining solution (3-5% methyl green dissolved in 0.1M barbituric acid PH4.0), please wash the excess methyl green with sterile distilled water. Then, it is washed (100% ethanol), dehydrated (xylene) transparent, and mounted after observing with an optical microscope. If 80-90% ethanol is used for washing at this time, methyl green is relatively easy to decolor, and pay attention to rapid dehydration.

7. The fluorescein-labeled dUTP solution contains carcinogens such as methylarsenate and cobalt dichloride, and can enter the body through inhalation, oral administration, etc., and pay attention to protection.

8. Reagent storage; unopened kits are stored at -20 ° C (-15 ~ 25 ° C); once the converter-POD solution is thawed, it will be stored at 4 ° C (2 ~ 8 ° C) and stable for at least 6 m To avoid freezing again; after preparing the TUNEL reaction solution before use, put it on ice until use.

9. Pay attention to the analysis of the results: a large number of DN ** breaks can be produced in the late stage of necrosis or in highly proliferated / metabolized tissue cells, thereby causing false positive results; and some types of apoptotic cell death lack DNA breaks or DNA Incomplete lysis and matrix components outside the cell prevent TdT from entering the intracellular reaction, resulting in false negative results

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