The study of the DNA damage response is necessary in understanding the pathophysiology of ovarian cancer potential biomarkers in novel targeted therapy. This technique allows for the sample to stay intact and allows the examination of antigens in their location of action. This protocol is quick and can be adapted to any antigen amenable to immunofluorescence.
This method gives insight into ovarian cancer and DNA damage repair processes involved in chemotherapeutic and PARP inhibitor resistance. These methods can be applied to other areas of study involving the use of 3D cultures and organoids. To begin, aspirate the media from the irradiated and incubated organoids without disrupting the 3D matrix.
Then wash with 300 microliters of PBS. Fix the organoids in 300 microliters of 2%paraformaldehyde for 10 minutes. After fixing, wash the organoids with 300 microliters of staining buffer and place them on a shaker for five minutes.
Then permeabilize the organoid by gently adding 300 microliters of permeabilization buffer. After 20 minutes of incubation, remove the buffer and wash with 300 microliters of staining buffer. Place the organoids on a shaker for five minutes.
Next, add 300 microliters of staining buffer to block the permeabilization step. Place on the shaker for 30 minutes and aspirate the staining buffer using a pipette. Then add 300 microliters of primary antibodies diluted in staining buffer.
Incubate at four degrees Celsius for 16 hours. After incubation, remove the primary antibody solution. Perform three washes with 300 microliters of staining buffer for five minutes each on the shaker.
Add 300 microliters of secondary antibody solution diluted in staining buffer, and incubate for one hour in the dark. After one hour, aspirate the secondary antibody solution and add 300 microliters of diluted DAPI. Place on the shaker for five minutes.
Perform three washes with 300 microliters of staining buffer for five minutes each on the shaker. Remove the staining buffer and detach the chambers using the removal kit included with the chamber slides. Cut the tip of a P200 pipette tip and use this to add mounting medium to cover each well containing an organoid tab.
Place a cover slip over the specimen while avoiding bubbles. Take clear nail polish and paint it onto the sides of the cover glass to seal the slide. Let it harden for one hour and then place it at 20 degrees Celsius.
Acquire images using a confocal microscope using a 63X objective with immersion oil. Using this protocol, patient derived ovarian cancer organoids were stained for DNA damage repair proteins before and after irradiation, and evaluated for biomarkers such as gamma H2AX, a marker of DNA damage, and RAD51, a marker for homologous recombination repair. RPA, a marker of replication stress, 53BP1, a marker of non-homologous enjoining, and Geminin, a GS2 phase cell cycle marker were also evaluated using this technique.
The BME tabs tend to depolymerize, so paying close attention after fixing and aspirating is vital. Analyzing the antigens to determine the organoids damage and the ability to mount a DNA damage response could help assist in the evaluation of chemotherapeutic resistance.
