Tunel and immunofluorescence

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TUNEL AND IMMUNOFLUORESCENCE

Plate 2  10⁵ cells onto a p60.
The following day transfect gene of interest along with the appropriate positive and negative controls.
Make sure there is one dish of untransfected cells for DNAse treatment.
Coat coverslips(Fisher brand Microscope cover glass) with a 1:10 solution of poly-L-lysine in sterile dH2O. Add 2 mls of solution per cover slip placed in the wells of a 6-well polystyrene dish. Incubate at RT for 20 minutes. Wash 3 times with sterile PBS. Aspirate the PBS and place the dish uncovered under UV for 5 minutes. Let the cover slips dry.
4 hours after transfection, split cells and plate them onto the individual prepared cover slips.
24 hours later, aspirate off the medium and wash the cells with PBS.
Fix the cells with 1ml of 4% paraformaldehyde(dilute 16% solution of paraformaldehyde[EMS cat#15710] with PBS) for 20 min at 4c.
Remove fixing solution and wash cells 2 times in PBS.
Aspirate PBS and add 1 ml of 0.2% Triton-X 100 in PBS.
Incubate 5 min at RT.
Remove permeabilising solution and wash cells twice with PBS.

For TUNEL staining:

Place 100 l of DNAse buffer onto a strip of parafilm placed on a flat surface. Invert the cover slip with untransfected cells(DNAse control) onto the DNAse buffer so that the cell side of the coverslip is facing the solution. Make sure the entire surface of the cover slip is coated with the solution(do not let the cells dry out at any time). Incubate for 10 min at RT.
Meanwhile prepare 100 l of fresh DNAse buffer containing 1 l of DNAse and place onto a clean spot on the same parafilm. Now place the coverslip on the DNAse containing solution in the same way as described above. Incubate for 15 min at RT.
Stop DNAse treatment by placing the coverslip back into the well (cell side up) and washing several times with dH2O.
Spot equilibration buffer (from PROMEGA kit, cat #:G3250 ) onto a clean strip of parafilm placed on a flat surface (100 l for each coverslip to be treated).
Incubate 5 min at RT.

Make enough TUNEL reaction mix for all the coverslips to be labelled. Each reaction is made up as follows:

Equilibration buffer:45 l

Nucleotide mix : 5 l

TUNEL enzyme : 1 l

18. Prepare a humidified chamber by placing a few wet paper towels in a plastic container with a lid.

Spot the reaction mix onto a clean strip of parafilm placed on the wet paper towels (50 l for each coverslip to be treated) and incubate in the dark(by wrapping the plastic container in aluminum foil) at 37 c, for 1 hour.
Stop TUNEL reaction by placing the coverslips back into the wells of the 6-well dish containing 2ml of 2X SSC. Incubating for 20 min at RT.
Wash cells several times with PBS.

For double staining (IMMUNOHISTOCHEMISTRY):

Block cells by adding 1ml of 10% goat serum in PBS to TUNEL treated cells.
Incubate in the dark at RT for 20 min.
Meanwhile spot the antibody solution(appropriate antibody diluted in PBS containing 1% goat serum)onto a clean strip of parafilm.
Remove blocking solution from wells and invert coverslips cell side down on the antibody solution.
Incubate CVs at RT for 1 hour in the dark.
Wash cells with PBS 3 times for 5 min each.
In a similar manner treat cells with fluorescent secondary antibody and incubate for 30 min at RT.
Wash cells with PBS 3 times for 5 min each.
Place 2ml of DAPI solution(1:1000 in PBS) in a clean well of the 6-well dish. Immerse indivisual cover slips one at a time for less than 5 seconds(just swirl quickly in the solution) in the well and replace the coverslips into the PBS.
Mount cells.
Spot Glass mounting slides(Fisher brand cat # 12-550-12) with mounting gel (Biomeda cat # M1.
Place coverslips cell side down the gel spots making sure the gel spreads evenly without bubbles.
Let dry for a few minutes and then seal the slide by brushing nail polish around it.
View under the microscope.

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