To begin, take cultured U2OS cells with 60%to 70%confluency and split them using 0.25%Trypsin-EDTA. Prepare 60 millimeter plates by adding round 13 millimeter cover slips. Plate 400, 000 cells in a 60 millimeter plate containing multiple 13 millimeter round cover slips.
Then, remove the culture medium from the plates. Add culture medium containing 25 micromolar etoposide, and incubate cells for 20 minutes, 1 hour and 3 hours. After incubation, remove the medium from the plates.
Wash the cells with PBS three times. To fix the cells, add ice cold methanol in enough volume to cover the cover slip surface, and incubate for 10 minutes at 20 degrees Celsius. Then, wash the cover slips with PBS three times and transfer them to a humidity chamber.
After tapping off PBS, add 30 to 40 microliters of permeabilization buffer and incubate for 20 minutes at room temperature. After that, wash the cover slips three times with PBS. Then, tap off the PBS from the samples.
Add 30 to 40 microliters of blocking solution provided in the PLA kit to each cover slip. Incubate the plate in a preheated humidity chamber at 37 degrees Celsius for 1 hour. Now dilute the primary antibodies in the antibody diluent provided in the kit.
After tapping off the blocking solution, add the primary antibody solution to each cover slip and incubate at four degrees Celsius overnight in a humidity chamber. Dilute the minus and plus probes one to five in the antibody diluent. Use combinations like Donkey anti-Mouse MINUS with Donkey anti-Goat PLUS and Donkey anti-Mouse MINUS with Donkey anti-Rabbit PLUS.
Now, tap off the primary antibody solution and wash once with TBST. After tapping off the excess TBST, add 20 microliters of the PLA probe solution to the cover slips. Then, incubate in the preheated humidity chamber at 37 degrees Celsius for 1 hour.
Dilute the 5x ligation buffer in high purity water. Tap off the PLA probe solution and wash once with TBST. Now, add ligase to the ligation solution at 1:40 dilution immediately before applying it to the samples.
Incubate in the preheated humidity chamber at 37 degrees Celsius for 30 minutes. Next, dilute the 5x amplification buffer in high purity water. Once the ligation solution is removed from the samples, wash the samples once with TBST.
Add the polymerase to the amplification solution at 1:80 dilutions immediately before applying it to the samples. Incubate in the preheated humidity chamber at 37 degrees Celsius for 100 minutes. Tap off the amplification buffer, then wash two times with SSC buffer for 10 minutes each.
After washing the samples two times with PBS, add the primary antibody solution to each cover slip and incubate at room temperature for 1 hour. At the end of the incubation, tap off the primary antibody solution and wash three times with TBST. Then, add the secondary antibody solution to each cover slip and incubate at room temperature for 20 minutes.
Afterward, wash five times with TBST, two times with SSC buffer, and two times with 0.01x SSC buffer. Finally, acquire images from different wells or cover slips regions to avoid artifacts due to non homogeneous incubation, and save all the channels with the same name pattern.
