DNA damage assessment in the adult Drosophila brain via pH2Av (S137) immunostaining

Fix flies of the desired genotype in 4% formalin, embedded heads in paraffin, cut

4-micron sections, and dry at 42˚C

Deparaffinize and bring through ethanol to water (xylene x 2, 100% ethanol x 2, 95% ethanol x 2, H2O x 2)

Microwave slides in 10 mM sodium citrate for 0h 15m 0s. Cool for 0h 20m 0s.

Stock: 100 mM sodium citrate, pH 6.0. Use at least 1L of citrate solution in a large glass box to avoid drying

Block in blocking solution (PBS with 0.3% Triton and 2% dry milk) for 30min to 1h 0m 0s

Incubate with primary antibody (Rockland pH2AV, cat # 600-401-914, dilution 1:100) overnight at room temperature

Wash 3 x in PBST (PBS with 0.3% Triton)

Incubate with fluorophore-coupled secondary antibody (Thermo Fisher) diluted 1:200 in blocking solution at room temperature

Wash 2 x in PBST and 1 x in PBS

Mount in medium containing DAPI (Southern Biotech)