TUNEL apoptosis detection kit instructions

Description: TUNEL (TdT mediated dUTP Nick End Labeling) is a classic method for detecting apoptosis. The kit uses the TUNEL method to catalyze the incorporation of fluorescent DNA at the 3'-hydroxy (3'-OH) end of the apoptotic cell rupture using a highly active recombinant DNA deoxynucleotidyl transferase (TdT). Apoptosis was detected by detecting FITC-12-dUTP-labeled DNA fragments by fluorescein-12-deoxytriphosphate uridine (FITC-12-dUTP). FITC-12-dUTP-labeled DNA can be directly observed with a fluorescence microscope or by flow cytometry. The kit has been optimized several times, and the FITC-12-dUTP-labeled and unlabeled dNTPs are in optimal matching ratio, so that when the 3'-OH terminal nucleotide is incorporated, the same fragmented DNA fragment can have more ends. The incorporation of FITC-12-dUTP greatly increases the sensitivity of the assay and reduces the background response. Together with our highly active TdT enzyme, the detection signal-to-noise ratio of this kit is greatly improved, and the background is very clean.

TUNEL FITC End-Fluorescent Labeling Apoptosis Detection Kit Features:

The ratio of labeled dNTP to unlabeled dNTP is optimized multiple times, high signal to noise ratio, and high sensitivity;
TdT enzyme is a genetically engineered recombinant product with high activity, high fluorescence incorporation efficiency and low background;
Suitable for smear, climbing, paraffin sectioning and frozen sectioning.
Kit Pack ~

product composition

Method steps:

Sample pretreatment scheme

• Paraffin-embedded tissue section
• Paraffin tissue sections were placed in xylene for 5 minutes at room temperature. Replace the new xylene and soak for 5 minutes to completely remove the paraffin;
• Soak for 5 minutes with 100% ethanol, replace with new 100% ethanol and soak for 5 minutes;

• Note: For best results, proteinase K incubation time may need to be optimized.

Tissue frozen section

• Immerse the fragment in 4% poly-methyl solution (dissolved in PBS) and incubate for 15 minutes at room temperature;
• Remove excess liquid and carefully blot the liquid around the sample on the slide with filter paper;
• The slide was immersed in PBS solution and incubated for 15 minutes at room temperature;
• Remove excess liquid and carefully blot the liquid around the sample on the slide with filter paper. A contour of the sample distribution is drawn around the sample with a paraffin pen or nail polish to facilitate downstream permeable processing and balanced marking operations. Do not allow the sample to dry during the experiment. The treated sample is placed in a wet box to keep the sample moist;
• 2 mg/ml proteinase K solution was diluted with PBS to a final concentration of 20 μg/ml;
• 100 μl of diluted Proteinase K solution was added dropwise to each sample, which was completely covered and incubated for 10 minutes at room temperature;
• Immerse the sample in the open beaker containing the PBS solution 2-3 times;
• Remove excess liquid and carefully blot the liquid around the sample on the slide with filter paper. The treated sample is placed in a wet box to keep the sample moist.

Cell sheet

• The cells were fixed, and the cell sheets were immersed in a dyeing cylinder containing 4% fresh paraformaldehyde in PBS, and allowed to stand at 4 ° C for 25 minutes;
• The slides were washed, immersed in PBS and allowed to stand at room temperature for 5 minutes. Repeat washing once with PBS;
• Gently remove excess liquid and carefully blot the liquid around the sample on the slide with filter paper. At this time, a paraffin pen or nail polish can be used to draw the outline of the sample distribution around the sample, facilitating downstream permeable processing and balance marking operation. Do not allow the sample to dry during the experiment. The processed sample is placed in the mixing box to keep the samples moist;
• The 2 mg/ml proteinase K solution was diluted with PBS to a final concentration of 20 μg/ml, and 100 μl of proteinase K solution was required for each sample;
• Add 100 μl of diluted proteinase K solution to each sample, cover it completely, and incubate for 5 minutes at room temperature (can also be immersed in 0.2% Triton® X-100 solution in PBS, incubated for 5 minutes at room temperature for transparency deal with);
• Immerse the sample in the open beaker containing the PBS solution 2-3 times;
• Gently remove excess liquid and carefully blot the liquid around the sample on the slide with filter paper. The treated sample is placed in a wet box to keep the sample moist.

Marking and testing

Dilute the 5× Equilibration Buffer to 1×Equilibration Buffer with deionized water.

Add 100ul of 1×Equilibration Buffer to cover the area of ​​the sample to be tested and incubate for 10-30 minutes at room temperature. At the same time, FITC-12-dUTP Labeling Mix was thawed on ice and a sufficient amount of TdT incubation buffer for all experimental and positive control reactions was prepared according to Table 1. For a standard reaction with an area of ​​less than 5 cm2, the volume is 50 [mu]l and 50 [mu]l is multiplied by the number of experimental and positive control reactions to determine the total volume of the desired TdT incubation buffer. For samples with larger surface areas, the reagent volume can be increased proportionally.

3) Use a blotting paper around the equilibrated area to remove excess Equilibration Buffer, do not let the tissue or cells dry, and then add 50 ul of TdT Incubation Buffer to the tissue or cells.

4) Cover the plastic coverslip on the cells to ensure the average distribution of the reagents. Place a paper towel soaked in water on the bottom of the wet box, place the slide in the wet box, and wrap the wet box with aluminum foil. Avoid light and incubate for 60 minutes at 37 °C.

Table 1. TdT incubation buffers prepared for experimental and positive control reactions

• Component	Volume (μl/50μl system)	Number of samples (experiment + positive control number)	total capacity (μl)
  Dd H2O	34
  5×Equilibration Buffer	10
  FITC-12-dUTP Labeling Mix	5
  Recombinant TdT Enzyme	1

  5) Remove the coverslip and incubate the sections in PBS solution for 5 minutes at room temperature.

• 6) Gently remove excess fluid and incubate for 5 minutes at room temperature with fresh PBS solution. Repeat this step once.

• 7) Use a filter paper to gently wipe off the PBS solution around the sample and back.

• Note: In order to reduce the background, you can try to wash the slide with PBS in step 5, then wash it three times with PBS containing 0.1% Triton® X-1 00 and 5 mg/ml BSA (instead of step 6) for 5 minutes each time. Thus, the free unreacted label can be cleared clean.

• 8) Immerse the slide in a dyeing cylinder containing the PI solution and leave it in the dark room for 5 minutes at room temperature. The PI solution was diluted to 1 μg/ml with PBS.
  9) Wash the sample, immerse the slide in deionized water, leave it at room temperature for 5 minutes, and wash it three times.
  10) Drain the excess water from the slide, but keep the tissue or cells moist.

• 11) Immediately analyze the sample under a fluorescence microscope and observe the green fluorescence at 520 ± 20 nm with a standard fluorescent filter. The red fluorescence of PI was observed at >620 nm. If necessary, the slides can be stored overnight at 4 ° C in the dark.

• 3. Positive control preparation: If necessary, a positive control can be prepared as follows.

• 1) After the sample is pretreated, add 100 ul of DNase I buffer to the fixed cells and incubate for 5 minutes at room temperature;

  2) Lightly remove the liquid, add 100 μl of buffer containing 5.5-10 units/ml DNase I, and incubate for 10 minutes at room temperature;

  3) Remove the excess liquid by gently rubbing the slide and wash the slide thoroughly 3-4 times in a dyeing tank filled with deionized water.

• Note: A separate staining cylinder must be used for the positive control slide. Otherwise residual DNase I activity from the positive control slide may introduce a high background on the experimental slide.

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The sections were each dipped once with gradient ethanol (90, 80, 70%) for 3 minutes each time, gradually increasing the moisture;

The sections were gently rinsed with PBS, and the excess liquid around the sample on the slide was blotted with filter paper. At this time, a paraffin pen or a hydrophobic pen can be used to draw the outline of the sample distribution around the sample, facilitating downstream permeable processing and balance marking operation. Do not allow the sample to dry during the experiment. The treated sample is placed in a wet box to keep the sample moist;

2 mg / ml of proteinase K solution was diluted with PBS to a final concentration of 20 μg / ml;

100 μl of the diluted proteinase K solution was added dropwise to each sample, and it was completely covered and incubated at room temperature for 20 minutes;

The sample was rinsed with PBS solution. Gently remove excess liquid and carefully blot the liquid around the sample on the slide with filter paper. The treated sample is placed in a wet box to keep the sample moist.

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