In the detection of programmed cell death, the literature shows by using Promega PCD kit, the TUNEL staining shows green fluorescence and DAPI staining shows blue fluorescence. I want to make it a little bit clear, should separate samples be prepared for TUNEL staining and DAPI staining, or the same sample should be first stained with TUNEL and then with DAPI and visualized under fluorescence microscope?
Hi there, DAPI staining is used for living cells and specifically binds to the Adenine and Thymine base locations on the natural DNA, but TUNEL staining is a method for detecting apoptotic cells that have fragmented DNA; this stain is attached to DNA fragment terminals.@
Hello Muhammad Husnain Ahmad
The same sample should be first stained with TUNEL and then with DAPI and visualized under fluorescence microscope. TUNEL staining can detect DNA degradation through the enzyme terminal deoxynucleotidyl transferase (TdT) to incorporate labeled dUTP into free 3′-hydroxyl termini created during the fragmentation of genomic DNA, which is a salient feature of PCD, while DAPI staining can reveal the nuclei of living or dead cells by emitting blue fluorescence upon binding to AT regions of DNA.
So, TUNEL assay will show a green dot if degraded DNA within a nucleus is detected, while DAPI staining will display a blue dot if a nucleus is detected.
Best.
Malcolm Nobre Thank you for your detailed answer. Can you please guide me about the fluorescence microscope settings? The protocol says we should use DAPI filter in the fluorescence microscope, but it only shows the blue fluorescence. So, should we change the filter to visualize the blue fluorescence for DAPI and green fluorescence for TUNEL?
In addition to the answers of Malcolm Nobre and Muhammad Husnain Ahmad, yes! You can change the filter to visualize the blue fluorescence for DAPI and the green fluorescence for TUNEL sequential acquisition after that, which superimposes the image.
Rajan Singh Thank you for your answer. So, in fluorescence microscope there is an option for DAPI filter, but, for green fluorescence, can we use GFP filter?
Muhammad Husnain Ahmad, yes for green fluorescence, you may use GFP filter. I also agree with Rajan Singh
The following filters may be used for examination of samples:
DAPI filter (excitation 340 – 390 nm, emission 420 – 470 nm)
GFP filter (excitation 450 – 490 nm, emission 500 – 550 nm)
Best.
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