TUNEL Assay Kit - FITC (ab66108)

The laboratory responded to your inquiry regarding visibility of a pellet after adding the washing buffer. You should be able to see at least a mini-speck of the pellet even after the wash step. One easy thing to do would be to take an aliquot of the cells in the second step of washing and look at it under the microscope. If you are able to see the cells, then they are there.

We have not tried this protocol on tissue sections, but here is a protocol that the lab has found from another which can be modified to fit into this procedure. A. Tissue Section Preparations: The protocol describes the preparation of formalin-fixed, paraffin-embedded tissue section mounted on glass slides. For information on fixing and embedding techniques, see Ben-Sasson et al., (Methods Cell. Biol. 46:29-39, 1995). Most steps are performed in Coplin Jars. Note: If using fresh-frozen tissue sections, proceed directly to step 7. 1. Remove paraffin by immersing slides in a Coplin jar containing fresh xylene. Incubate at room temperature for 5 minutes. 2. Repeat in a second Coplin jar containing fresh xylene. 3. Immerse the slides in a Coplin Jar containing 100% ethanol and incubate at room temperature for 5 min. 4. Rehydrate the slides by sequential 3-min, room temperature incubations in Coplin jars containing: · 100% ethanol · 95% ethanol · 85% ethanol · 70% ethanol · 50% ethanol 5. Immerse the slides in a Coplin jar containing 0.85% NaCl and incubate at room temperature for 5 min. 6. Immerse the slides in a Coplin jar containing PBS and incubate at room temperature for 5 minutes. 7. Fix the slides by immersing them in a Coplin jar containing fresh 4% formaldehyde/PBS, and incubate at room temperature for 15 min. 8. Wash the slides by immersing them in a Coplin jar containing PBS, and incubate at room temp. for 5 min. 9. Transfer to another Coplin jar containing PBS, and incubate at room temperature for 5 min. 10. Allow the liquid to drain thoroughly and place slides on a flat surface. 11. Prepare 20 µg/ml of Proteinase K Solution (combine 2 µl of 10 mg/ml Protease K and 998 ml of 100 mM Tris-HCl, pH 8.0, 50 mM EDTA) and cover each section with 100 µl of it. Incubate at room temperature for 5 min. 12. Immerse the slides in Coplin jar containing PBS, and incubate at room temperature for 5 min. 13. Transfer the slides to a Coplin jar containing 4% formaldehyde/PBS and incubate at room temperature for 5 minutes. 14. Wash the slides by immersion in Coplin jar containing PBS, and incubate at room temperature for 5 min. B. Detection by Fluorescence Microscopy: 1. Remove slides from PBS and tap gently to remove excess liquid. Cover the cells in 100 µl of Wash buffer (blue cap). 2. Use forceps, gently place a piece of plastic coverslip on top of the cells to evenly spread the liquid, incubate for 5 min. Remove plastic coverslip and gently tap the slides to remove excess liquid. 3. Repeat step 2. Carefully blot dry around the edges with tissue paper. 4. Gently place 50 µl of the DNA Labeling Solution (prepared as in Section IIIB, Step 4) on the cells. 5. Use forceps, gently place a piece of plastic coverslip on top of the cells to evenly spread the liquid. 6. Place the slides in a dark, humidified 37°C incubator for 60 minutes. Note: Ensure high humidity by placing wet paper towels in the bottom of the dry incubator. Antibody Solution 1 assay 10 assays Anti-BrdU-FITC Antibody (orange cap) 5 µl 50 µl Rinse Buffer (red cap) 95 µl 950 µl 7. Using forceps, remove the plastic coverslips. Rinse the slides to a fresh Coplin jar filled with PBS for 5 min. 8. Repeat step 7. Carefully blot dry around the edges with tissue paper. 9. Place 100 µl of the Antibody Solution (Prepared as in Section IIIB, step 8). 10. Use forceps, gently place a piece of plastic coverslip on top of the cells to evenly spread the liquid. 11. Incubate the cells with the antibody solution in a humidified incubator for 30 min at room temperature. 12. Carefully remove the solution from slides. Add 100 µl of Propidium Iodide/Rnase A solution (amber bottle). 13. Use forceps; gently place a piece of plastic coverslip on top of the cells to evenly spread the liquid. 14. Incubate the slides in the dark in a humidified incubator for 30 min at room temperature. 15. Wash the cells by transferring the slides to a fresh Coplin jar filled with ddH2O and incubate at room temperature for 5 min. 16. Repeat Step 15. 17. [Optional] Add a drop of anti-Fade solution and cover the treated portion of the slide with a glass coverslip. 18. [Optional] Seal the edges of the coverslip with rubber cement or clear nail polish. 19. View slides as soon as possible using FITC and rhodamine filters. Apoptotic cells will exhibit strong nuclear green fluorescence. All cells should be stained with PI and exhibit strong red counter staining.

Thank you for your inquiry and your patience while we looked into this further.

For ab66108, the kit components are optimized for usage on cells. If using tissue sections, you will not be able to get a full 50 assays from the kit because you will run short of reaction buffer.
Here’s the protocol for using ab66108 for paraffin embedded tissue sections:
DEPARAFFINIZATION AND REHYDRATION
1. Immerse slides in xylene (or xylene substitute) for 5 minutes at room temperature. Repeat using fresh xylene for second 5 minute incubation.
2. Immerse slides in 100% ethanol for 5 minutes at room temperature. Repeat using fresh 100% ethanol for second 5 minute incubation.
3. Immerse slides in 90% ethanol for 3 minutes at room temperature.
4. Immerse slides in 80% ethanol for 3 minutes at room temperature.
5. Immerse slides in 70% ethanol for 3 minutes at room temperature.
6. Immerse slides briefly into 1X PBS and carefully dry the glass slide around the specimen.
Do not let the tissue specimen dry out during or between any step!
(If necessary, cover or immerse the specimen in 1X PBS to keep hydrated)
** At this point it may be helpful to encircle the specimen using a waxed pen or a hydrophobic marker.
PERMEABILIZATION OF SPECIMEN
1. Dilute enough Proteinase K (STOCK: 2 mg/ml in 10 mM Tris pH 8) needed 1:100 in 10 mM Tris pH 8.
2. Cover the entire specimen with 100 μl diluted proteinase K. Incubate at room temperature for 20 minutes. DO NOT OVER INCUBATE.
3. Rinse slide with 1X PBS.
4. Gently tap off excess liquid and carefully dry the glass slide around the specimen.
EQUILIBRATION AND Staining REACTION
1. Dilute Reaction Buffer (green cap) as needed 1:5 with dH2O.
(NOTE: This buffer does NOT contain nucleotides or enzyme).
2. Cover the entire specimen with 100 μl of the diluted Reaction Buffer. Incubate at room temperature for 10 to 30 minutes while preparing the staining reaction mixture.
3. For making staining solution, follow K402-100 kit protocol.
4. Carefully blot the Reaction Buffer from the specimen, taking care not to
touch the specimen.
5. Immediately apply 50 μl of staining solution onto each specimen.
** The use of a cover slip at this point assures even distribution of the reaction mixture and prevents loss due to evaporation during incubation. **
6. Cover the specimen with a piece of Parafilm cut slightly larger than the specimen. HINT: Folding up one corner of the Parafilm cover slip will aid in its application and removal.
7. Place slides in a humid chamber and incubate at 37C for 1 to 1.5 hours Note: incubation times at 37C may need to be adjusted to longer or shorter periods depending on the characteristics of the tissue supplied by the researcher..
8. Remove Parafilm cover slip and rinse slide with 1X PBS.
9. Repeat the rinsing step.
10. Gently tap off excess liquid and carefully dry the glass around the specimen.
11. Dilute PI/RNase staining buffer 1:5 in PBS & apply 50 ul of diluted Propidium Iodide/RNase A Solution (amber bottle) onto the specimen.
12. Incubate in the dark for 30 min at room temperature.
13. Analyze by fluorescence microscopy (apoptotic cells show green staining over an orange-red PI counter-staining)




I hope this information helps. Please contact us with any other questions.

Thank you for contacting us. This kit will work fine with the samples you describe.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you for your enquiry and your interest in our products. The two kits (ab66108 and ab66110) are mainly used for cells analyzed by flow cytometry. Currently, we do not have specific kits for tissue samples (for DNA Fragmentation) but I would advise you and your customers to check our catalogue regularly since we add and publish new products every day. Apologies! If you need any further assistance in the future, please do not hesitate to contact me.