Jan 23, 2024

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

DOI
dx.doi.org/10.17504/protocols.io.dm6gp36q5vzp/v1
  • 1Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Beatrice Weykopf
Open access
DOI: dx.doi.org/10.17504/protocols.io.dm6gp36q5vzp/v1
Protocol Citation: Mel Feany 2024. DNA damage assessment in the adult Drosophila brain via pH2Av (S137) immunostaining. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp36q5vzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 17, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 93685
Keywords: ASAPCRN
Abstract
This protocol describes how to stain adult Drosophila brains with pH2Av to assess DNA damage.
Fly fixation and paraffin embedding
Fly fixation and paraffin embedding
Fix flies of the desired genotype DurationOvernight in 4% formalin, embedded heads in paraffin, cut
4-micron sections, and dry at 42˚C DurationOvernight

Deparaffinization
Deparaffinization
Deparaffinize and bring through ethanol to water (xylene x 2, 100% ethanol x 2, 95% ethanol x 2, H2O x 2)
Antigen-retrieval
Antigen-retrieval
35m
Microwave slides in 10 mM sodium citrate for Duration00:15:00 . Cool for Duration00:20:00 .
Stock: 100 mM sodium citrate, pH 6.0. Use at least Amount1 L of citrate solution in a large glass box to avoid drying

35m
Blocking
Blocking
1h 30m
Block in blocking solution (PBS with 0.3% Triton and 2% dry milk) for 30min to Duration01:00:00
1h
Antibody incubation
Antibody incubation
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
Mounting
Mounting
Mount in medium containing DAPI (Southern Biotech)