Anyone have experience with TUNEL assay analysis?

Hi Juliet, it seems to be a crosstalk between the green and the Dapi channel. The reason: autofluoresscence of the muscle tissue which would be enhanced after paraffin embedding. I recommand to use a orange (Fluor 555) or darkred Fluophore (Fluor 647) .Here are more informations: https://www.elabscience.com/List-detail-459246.html; And you should use a positive control of proliferating tissue like gut or liver to check wether your kit works specifically.

Saman Behboodi Tanourlouee

Thanks for providing a detailed description of your procedure, it really helps in troubleshooting the issue. It sounds like you're having difficulty distinguishing between the DAPI-labeled nuclei and the FITC-labeled apoptotic nuclei in your TUNEL assays. Here are a few possible explanations and potential solutions:

Specificity of TUNEL Staining: TUNEL staining can sometimes produce non-specific results and can even label healthy cells, especially if the tissues are not optimally preserved. If the protocol is not carefully optimized, you might see the TUNEL signal in all nuclei, which would make it look similar to the DAPI stain. It's also possible that there is a high degree of apoptosis in your samples that you're not expecting, causing most of the nuclei to be labeled.

Fluorescence Bleed-through: If there's spectral overlap between the DAPI and FITC channels, you might be picking up signal from one in the other. Ensure that the filters on your microscope are appropriately set for each fluorophore to avoid this.

Fixation and Permeabilization: Over-fixation can mask the TUNEL signal, while under-fixation can lead to excessive staining. Similarly, incorrect permeabilization can prevent the TUNEL reagents from reaching the DNA or can cause excessive staining. You might need to optimize your fixation and permeabilization steps.

Camera Settings: Overexposure can also cause issues with distinguishing between signals. Check your exposure settings and adjust them to ensure you're not overexposing the FITC channel.

Positive and Negative Controls: Use positive and negative controls to ensure the assay is working properly. For the positive control, you can treat cells with a DNAse I to induce DNA breaks and should see a strong TUNEL signal. For the negative control, perform the assay without the terminal transferase enzyme to confirm that the signal you're seeing is specific. Try to systematically rule out each potential issue. If you still can't resolve the issue, you might need to consider using a different assay for detecting apoptosis, such as Annexin V/PI staining or caspase activity assays. Good luck!

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