5. Click-iT 488 Tunel Cell Apoptosis Detection Kit; 50 T $776

Step 1: Sample Preparation

A. Paraffin Embedding Tissue Sections

1. Immerse paraffin tissue sections in xylene at room temperature for 5-10 minutes, repeat 2-3 times. Then immerse in absolute ethanol for 5 minutes, repeat 2 times. Finally, immerse in gradient ethanol (85%, 75%, double-distilled water) for 1 time each, 5 minutes each time.
2. Rinse the sections gently with PBS and remove excess liquid around the samples. Use a histology pen to draw a small circle about 2-3 mm away from the tissue perimeter, which facilitates subsequent permeabilization and labeling steps. Throughout the experiment, make sure the samples do not dry out. Keep the processed samples in a humid chamber.
3. Prepare Proteinase K working solution: Dilute Proteinase K (200 µg/mL) stock solution in PBS with a ratio of 1:9, resulting in a final concentration of 20 μg/mL.
4. Add 100 μL of the Proteinase K working solution onto each sample, ensuring it covers the entire sample. Incubate at 37°C for 20 minutes.
5. Rinse the samples 3 times with PBS for 5 minutes each time to remove Proteinase K (failure to wash away Proteinase K may interfere with subsequent labeling reactions). Keep the processed samples in a humid chamber.
6. (Optional step) Remove excess liquid from the samples and add an appropriate amount of membrane permeabilization solution to fully infiltrate the tissue. Incubate at room temperature for 20 minutes. After membrane permeabilization, rinse the samples with PBS 3 times for 5 minutes each time. Keep the processed samples in a humid chamber.

B. Frozen Tissue Sections

1. Immerse frozen tissue sections in a fixation solution and incubate at room temperature for 10-15 minutes.
2. Remove the tissue sections from the fixation solution and air dry in a fume hood.
3. Rinse the tissue sections in distilled water or PBS to remove residual fixation solution.
4. Use a histology pen to draw a small circle about 2-3 mm away from the tissue perimeter. Throughout the experiment, make sure the samples do not dry out. Keep the processed samples in a humid chamber.
5. Prepare Proteinase K working solution: Dilute Proteinase K (200 µg/mL) stock solution in PBS with a ratio of 1:9, resulting in a final concentration of 20 μg/mL.
6. Add 100 μL of the Proteinase K working solution onto each sample, ensuring it covers the entire sample. Incubate at room temperature for 10 minutes.
7. Rinse the samples 2-3 times with PBS to remove excess liquid (Proteinase K should be thoroughly washed away to avoid interference with subsequent labeling reactions). Keep the processed samples in a humid chamber.
8. (Optional step) Add an appropriate amount of membrane permeabilization solution to fully infiltrate the tissue. Incubate at room temperature for 20 minutes. After membrane permeabilization, rinse the samples with PBS to remove excess liquid. Keep the processed samples in a humid chamber.

C. Cell Cytospin

1. Culture adherent cells in Lab-Tek Chamber Slides. After apoptosis induction treatment, rinse the chamber slides gently with PBS twice.
2. Add an appropriate amount of fixation solution to cover the tissue in each chamber slide. Incubate at room temperature for 20 minutes.
3. Remove the fixation solution and rinse with PBS 3 times for 5 minutes each time.

4. Step 4: Permeabilization

   1. Immerse each sample in a 0.2% Triton X-100 solution prepared in PBS, and incubate at room temperature for 5 minutes.
   2. Rinse the samples 2-3 times with PBS in an open beaker containing PBS solution.
   3. Gently remove excess liquid and use filter paper to carefully dry the area around the samples on the glass slide. Keep the processed samples in a humid chamber to maintain their moisture.

   Step 5: Permeabilization (continued)

   4. Repeat the permeabilization step by immersing each sample in a 0.2% Triton X-100 solution prepared in PBS at room temperature for 5 minutes.
   5. Rinse the samples 2-3 times with PBS in an open beaker containing PBS solution.
   6. Gently remove excess liquid and use filter paper to carefully dry the area around the samples on the glass slide. Keep the processed samples in a humid chamber to maintain their moisture.

   Step 2: DNase I Treatment Positive Control Experiment (Optional)

   After permeabilization of the samples, perform a positive control experiment by treating the samples with DNase I (recommended G3342).

   1. Add 100 μL of 1x DNase I Buffer (prepared by taking 10 μL of 10x DNase I Buffer and adding 90 μL of deionized water) onto the permeabilized samples and incubate at room temperature for 5 minutes.
   2. Gently remove excess liquid and add 100 μL of working solution containing DNase I (20 U/mL) (prepared by taking 10 μL of 10x DNase I Buffer, adding 2 μL of DNase I, and then adding 88 μL of deionized water) onto the samples. Incubate at room temperature for 10 minutes.
   3. Gently remove excess liquid and thoroughly wash the glass slide in a staining jar containing PBS, repeating the wash 3-4 times.

   Step 3: Labeling and Detection

   1. Equilibration: Add 50 μL of Equilibration Buffer onto each sample to cover the entire area of the sample. Incubate at room temperature for 10 minutes.
   2. Labeling solution preparation: Thaw EdUTP Labeling Mix and Equilibration Buffer on ice. Mix Recombinant TdT enzyme, EdUTP Labeling Mix, and Equilibration Buffer in a ratio of 1 μL:5 μL:50 μL (1:5:50) to prepare enough TdT incubation buffer for all experiments. Adjust the volume of reagents used in the experiment according to the size of the glass slide.
   3. Negative control system: Prepare a control TdT incubation buffer without Recombinant TdT enzyme, using ddH2O instead.
   4. Labeling: Remove excess Equilibration Buffer and add 56 μL of TdT incubation buffer onto each tissue sample. Incubate at 37°C for 1 hour. Avoid drying the slide.
   5. Immediately wash the tissue samples with PBS, repeating the wash 4 times for 5 minutes each time.
   6. Click reaction: Remove the previous PBS buffer and add 100 μL of Click reaction solution onto each sample, ensuring it covers the entire sample. Incubate at room temperature in the dark for 30 minutes. (Refer to the table below for the Click reaction solution composition, add each reagent while mixing, and adjust the preparation amount proportionally as needed. It is recommended to prepare in advance.)

   Component	Volume
   Reaction Buffer A	925 μL
   Reaction Buffer B	10 μL
   iF488 azide dye	15 μL
   Reaction additive (Reagent C)	50 μL
   Total volume	1000 μL

   7. Remove the click reaction solution and wash the sample with PBS buffer 2-3 times, each time for 5 minutes.
   8. Gently remove the PBS solution around the sample using a filter paper.
   9. Nuclear staining: Stain the sample in a staining dish by immersing the slide in a staining dish containing DAPI solution (prepared and diluted with fresh PBS) at room temperature for 8 minutes (or use Hoechst 33258 for nuclear staining).
   10. Mounting: After the sample staining is complete, wash the tissue sample with PBS 3 times, each time for 5 minutes. Then gently remove the excess liquid and add anti-fade mounting medium (recommended G1401) to mount the sample.
   11. Microscopic examination: Immediately analyze the sample under a fluorescence microscope. Handle the slide carefully and avoid light exposure. DAPI stains both apoptotic and non-apoptotic cells blue, while the iF488 azide dye specifically localizes in the apoptotic cell nuclei and emits green fluorescence.

   Four, Detection of Suspended Cells by Flow Cytometry

   1. Wash the cells to be analyzed twice with PBS, centrifuge at 4℃ (500 g), and resuspend in 500 µL of PBS.
   2. Fixation: Add 5 mL of 1% paraformaldehyde solution prepared in PBS to the sample, fix the cells, and place on ice for 20 minutes.
   3. Centrifuge the cells at 4℃, 300 g for 10 minutes, remove the supernatant, and resuspend in 5 mL of PBS twice. Finally, resuspend the cells in 500 µL of PBS.
   4. Permeabilization: Add PBS solution containing 0.2% Triton X-100 to the cell sample, incubate at room temperature for 5-10 minutes to permeabilize the cells.
   5. Centrifuge the cells at 300 g for 10 minutes, resuspend in 5 mL of PBS, and centrifuge again. Then, resuspend the cells in 1 mL of PBS.
   6. Equilibration: Transfer approximately 2×106 cells to a 1.5 mL microcentrifuge tube, centrifuge at 300 g for 10 minutes, remove the supernatant, and resuspend in 80 μL of Equilibration Buffer. Incubate at room temperature for 5 minutes.
   7. Prepare the labeling solution: Thaw the EdUTP Labeling Mix and Equilibration Buffer on ice. Mix Recombinant TdT enzyme, EdUTP Labeling Mix, and Equilibration Buffer in a ratio of 1 µL: 5 µL: 50 µL (1:5:50) to prepare enough TdT incubation buffer for all experiments and optional positive control reactions.
   8. Labeling: Centrifuge the cells at 300 g for 10 minutes, remove the supernatant, and resuspend the pellet in 56 μL of TdT incubation buffer. Incubate at 37℃ for 1 hour, avoiding light. Gently resuspend the cells every 15 minutes using a micropipette.
   9. After the reaction is complete, remove the labeling reaction solution and wash the sample with PBS, washing 3-4 times, each time for 5 minutes.
   10. Click reaction: Remove the previous PBS buffer and add 100 μL of click reaction solution to the sample, ensuring that it covers the entire sample. Incubate at room temperature, avoiding light, for 30 minutes. (Refer to the table below for the click reaction solution,

       Please note that some of the steps may require specific reagents and equipment, and it’s essential to follow the protocols and safety guidelines provided by your institution or the specific procedure you are following.add each reagent in sequence while mixing. The preparation quantity can be proportionally increased or decreased. It is recommended to prepare in advance.)

   Component	Volume
   Reaction Buffer A	925 μL
   Reaction Buffer B	10 μL
   iF488 azide dye	15 μL
   Reaction additive（Reagent C）	50 μL
   total valum	1000 μL

   11. Remove the click reaction solution and immediately wash with PBS buffer 2-3 times, each time for 5 minutes.
   12. Remove the supernatant and resuspend the pellet in 1 mL of PBS containing 0.1% Triton X-100, which includes 5 mg/mL BSA. Repeat the wash 2 times.
   13. Nuclear staining: Centrifuge at 300 g for 10 minutes, remove the supernatant, and resuspend the cell pellet in 0.5 mL of DAPI solution, which includes 250 μg of RNase A without DNAase. Incubate the cells at room temperature in the dark for 30 minutes (or use Hoechst 33258 for nuclear staining).
   14. Analysis: Analyze the cells using a flow cytometer. DAPI stains both apoptotic and non-apoptotic cells blue, while the iF488 azide dye specifically localizes in the apoptotic cell nuclei and emits green fluorescence.

   Figure 5: Overview of the experimental procedure. please send Email if you need. Thanks.

   Please note that the specific reagents, concentrations, and timings mentioned in the steps are essential for the procedure. It’s important to follow the protocols and safety guidelines provided by your institution or the specific procedure you are following.