To begin, culture the chopped cerebellar tissue retrieved from 10 day-old mouse pup in basal medium eagle. Add DNA damaging chemicals directly to the culture medium at appropriate final concentrations. After the exposure period, replace the medium with a freshly prepared mixed medium containing the DNA damaging chemicals and the poly ADP ribose glycohydrolase inhibitor, and incubate for 30 minutes.
After 30 minutes, wash the slices with 0.1 molar phosphate buffer at room temperature and immediately fix them with a pre-cooled acetone and methanol mixture. Incubate the plate for 20 minutes at minus 20 degrees Celsius. Then remove the fixation solution and wash it with 0.4 molar phosphate buffer.
Add 100 microliters of 0.1 molar phosphate buffer to each well of a 48 well plate. Then use a scalpel and a cutting board to remove the margins of the insert, cutting around the tissue slice. Place the slice in a well of the 48 well plate.
After aspirating the buffer, incubate the insert with 100 microliters of 0.1 molar phosphate buffer containing 1%Triton X100. Aspirate the solution and serially incubate the insert in each well on a shaker with appropriate amounts of blocking solution followed by the primary in the secondary antibodies. Add 20 microliters of the final DAPI solution to each well 20 minutes before the end of the two hour incubation with the secondary antibody and continue shaking for 20 minutes.
For mounting, place the tissue on a microscope glass slide with the tissue facing up. Add the mounting medium and cover with a cover glass. Place the slides on a flat surface and let them dry overnight in the dark at four degrees Celsius.
The cerebellar foliation was maintained in the culture. Purkinje cells were stained for Calbindin D-28K and neural nuclei stained for NeuN. The astrocytes in the Purkinje cells were stained for GFAP.
PAR staining increased in treated Purkinje cells after exposure to potassium bromate and paraquat indicating DNA damage.
