Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

The overall goal of this procedure is to detect DNA breaks and cell death in C two. This is accomplished by first perme tissue sections to allow tunnel reagents to enter the cell nucleus. Next TDT enzyme is used to covalently attach DTPs to three prime ends of DNA breaks in these cells.

Then the DUTP is tagged with a fluorescent probe. Ultimately, the tunnel positive signal is quantified in tissue sections reflecting the relative frequency of DNA breaks in the cells. The main advantage of this technique over existing methods like southern blotting or immunohistochemistry, is that it's relatively fast specific and provides a way to eliminate noise and autofluorescence.

Start by fixing isolated hind limbs in 4%Para formaldehyde in PBS in a 15 milliliter tube allow the fixation to go for four to at most 24 hours at four degrees Celsius. Only two hours are needed if the tissue is embryonic. However, after the fixation, replace the solution with 10%sucrose to cryo protect the tissue and incubate it overnight at four degrees Celsius.

The next day, change the solution to sterile filtered 30%sucrose and continue the incubation overnight or until the hind limbs sink to the bottom of the tube. When embedding, it's important that the hind limbs are completely submerged in the embedding medium. The embedding molds can be directly placed on dry ice or in isop pentane on dry ice to freeze the medium.

When cryosectioning. The blocks cut transfer sections that are 10 microns thick or thinner, thicker sections will not be penetrated by the tunnel labeling reagents. Collect these thin sections onto gelatin or vector bond coated glass slides at room temperature.

Very quickly, transfer the slides to dry ice for storage. First thaw a slide of frozen tissue section to room temperature and let it dry for 16 to 64 hours. Once the slide is at room temperature, rehydrate the section in a coupling jar filled with PBS for 10 minutes.

Repeat this rehydration step once, then carefully dry the slide with a lab wipe without disturbing the tissue section to permease the section, place the slide into a humidity chamber and pipette 50 microliters of non proteolytic saponin base commercial reagents onto the section. Do not pipette directly onto the sections as it can detach or be damaged. Then place a small piece of para film over the section to spread out the reagent.

If bubbles form, reapply the para film. Let the slide incubate for an hour after an hour. Remove the reagents using two five minute washes in PBS and carefully dab it dry.

Next to label DNA breaks by TDT, use a commercially available kit. First, make the one XTDT buffer and pipetted over the section. Then incubate the slide for five minutes in the humidify chamber.

Second, make a TDT labeling mix according to the kit instructions or a positive control slide. Add DNA to the TDT labeling mix. For a negative control slide, omit the TDT enzyme from the mix.

Now using a wipe, remove as much buffer as possible from the slide and pipette it on the labeling mix. 50 microliters should suffice. Again, spread the solution with paraform without making bubbles.

Let the slide incubate in the chamber for an hour now at 37 degrees celsius. Third, make the one x stop buffer according to the kit's instructions. After removing the labeling mix with the lab wipe, add between two and 300 microliters of the stop mix to completely cover the section.

Then incubate the slide for five minutes in the chamber. No para film is used in this step. To remove the stop buffer, use two two minute washes in PBS.

Now pipette 50 microliters of freshly prepared fluorescein labeling solution. Onto the slide, apply perfil and incubate for 20 minutes in the chamber, but now in the dark. Remove the labeling solution with a single two minute wash in PBS while washing dilute some hooks.

3 3, 2, 5 8 to one microgram per milliliter in PBS. After the washes, cover the sections in one to 200 microliters of the diluted hooks solution. Then incubate the slide for five minutes, shielded from light to remove the hooks to stain, use three five minute washes in PBS.

Then carefully dry the slide with a lab wipe and apply a cover slip for imaging. Image the section using dpi, fitzy and Texas red filters to separate the hooks stain, tunnel stain and autofluorescence signals into three false color channels in a single image. Standardize and maintain the same imaging settings between all the slides so that they can be easily compared.

Do not use any automatic exposure or gain settings for anatomical reference. Use a bright brightfield image of an adjacent hind limb section stained with h and e in the image analysis software. Turn off the tunnel channel.

Then manually trace the outline of the muscle area to be quantified using the other channels. Convert the traced area into a mask. Then turn the tunnel channel back on and adjust the minimum intensity threshold to exclude autofluorescent signal.

Use the same threshold limit on all the sections analyzed in the study. Next, convert the threshold pixels into a mask. In each image, combine the muscle area mask and the threshold tunnel mask.

This makes a representation of the tunnel signal within the traced muscle area. Now quantitate the combined mask to determine the number of tunnel positive objects and the total pixel area of the tunnel signal within muscle. Use these values for statistical analysis.

Tunnel positive objects that low magnification may appear as bright irregular fragments in skeletal muscle. However, at higher magnification, some tunnel positive objects with the classic apoptotic morphology should be observed if the cell death type involved this apoptosis. The positive control with D DNAs added should exhibit abundant tunnel positive signal distributed uniformly across all tissues.

In the section. The negative control without TDT enzyme should have a low intensity background and show only autofluorescence. With the described protocol, it was found that RBCs bone and endothelial cells contributed to the autofluorescent signal in the prepared high limb sections.

True tunnel positive signal was much more intense. It could be isolated from the autofluorescent signal using a pixel intensity threshold. Another option to isolate the tunnel signal was to digitally subtract the red autofluorescence channel from the tunnel channel.

Using this technique, skeletal muscle cell death. In a mouse model of SMA was assessed, tunnel labeling revealed more apoptotic cells in five day old SMA mice than in controls. By quantifying the tunnel staining, this observation was shown to be statistically significant for multiple muscle groups within the hind limb.

Once mastered, the tunnel technique can be done in about four hours to generate reliable quantitative results.