Immunohistochemistry protocol for γH2AX detection (formalin-fixed paraffin-embedded sections)
DNA damage accumulation and consequent DNA damage response activation may be the result of a variety of DNA insults and may contribute to the phenotypes associated with aging and cancer initiation. One of the most robust markers of the generation of a DNA double strand break and DNA damage response activation is the phosphorylation of the histone H2AX on serine 139. The modified form (also known as γH2AX) labels the sites of DNA damage by spreading for megabases of chromatinised DNA. Its detection is therefore a reliable readout of the cellular events associated with DNA damage generation.
We describe here a detailed protocol for the reliable detection of γH2AX in mammalian (mouse or human) formalin-fixed paraffin embedded tissues.
Formalin-fixed, paraffin-embedded tissue sections cut at 2μm.
Histolemon-Erba, Carlo Erba Reagents, cat. 454912
EDTA 0.25mM pH8
H2O2 30%, Sigma cat. H1009
Ab mix: TBS1X – BSA4% - Tween20 0.02%
γH2AX mouse Upstate Biotechnology (cat.05-636) 1:200 in Ab mix
anti-mouse DAKO Cytomation Envision+System Labelled Polymer-HRP, cat. K4001
DAKO Cytomation liquid DAB+ Substrate Chromogen System, cat. K3468
Harris Hematoxylin, EMS cat. 26041
Eukitt, O. Kindler GmbH&Co
1.Deparaffinize tissue sections in Histolemon 2X10 min
2.Hydrate tissue sections through graded alcohol series (100%,95%,70%, H2O) 1X5 min
3.Incubate the slides in EDTA for 50 min at 95°C for antigen unmasking
4.Cool the slides for 20 min at room temperature (RT), then wash once in H2O
7.Preincubate the slides with Ab mix for 20 min at RT
8.Incubate the slides ON at 4˚C with anti-γH2AX diluted in Ab mix at indicated concentration, then wash twice in TBS 1X
12.Dehydrate tissue sections through graded alcohol series and mount with Eukitt
13.Positive (irradiated skin) and negative (not irradiated skin) controls were included in each experiment
Approximately 5h
The specificity of the antibody in immunohistochemistry must be verified by testing known positive and negative controls. A positive control is the tissue in which the molecule of interest is known to be expressed and in the specific case a X-rays irradiated tissue. On the other side, the simplest negative control is the absence of expression in tissues in which the molecule of interest is known not to be expressed. A better negative control is the elimination of the signal by pre-incubating the antibody with an excess of the peptide or protein with which it was raised, or by replacing the primary antibody with buffer in Ab mix. Each single experiment should run positive as well as negative controls simultaneously with the testing samples.
Antigen unmasking can be optimized for different antibodies.
DAB solution must be prepared just before the use according to manufacturer’s protocol.
When tissue staining has not given the expected results, several experimental variables should be considered:
NO STAINING
1) Confirm that no reagents were omitted and that were added in the correct order and for sufficient incubation times.
2) Check antibody titrations and dilutions. This is particularly important for the primary antibody.
3) Check reagent expiration dates and storage.
4) Check specimen storage.
5) Check that the microscope is adjusted correctly.
6) Do not let the slides air dry in any step of the procedure
BACKGROUND
Immunocytochemical Methods and Protocols (second edition), edited by Lorette C. Javois, from Methods in Molecular Medicine, volume 115, Humana Press, 1999
Posted 06 Dec, 2006
Immunohistochemistry protocol for γH2AX detection (formalin-fixed paraffin-embedded sections)
Posted 06 Dec, 2006
DNA damage accumulation and consequent DNA damage response activation may be the result of a variety of DNA insults and may contribute to the phenotypes associated with aging and cancer initiation. One of the most robust markers of the generation of a DNA double strand break and DNA damage response activation is the phosphorylation of the histone H2AX on serine 139. The modified form (also known as γH2AX) labels the sites of DNA damage by spreading for megabases of chromatinised DNA. Its detection is therefore a reliable readout of the cellular events associated with DNA damage generation.
We describe here a detailed protocol for the reliable detection of γH2AX in mammalian (mouse or human) formalin-fixed paraffin embedded tissues.
Formalin-fixed, paraffin-embedded tissue sections cut at 2μm.
Histolemon-Erba, Carlo Erba Reagents, cat. 454912
EDTA 0.25mM pH8
H2O2 30%, Sigma cat. H1009
Ab mix: TBS1X – BSA4% - Tween20 0.02%
γH2AX mouse Upstate Biotechnology (cat.05-636) 1:200 in Ab mix
anti-mouse DAKO Cytomation Envision+System Labelled Polymer-HRP, cat. K4001
DAKO Cytomation liquid DAB+ Substrate Chromogen System, cat. K3468
Harris Hematoxylin, EMS cat. 26041
Eukitt, O. Kindler GmbH&Co
1.Deparaffinize tissue sections in Histolemon 2X10 min
2.Hydrate tissue sections through graded alcohol series (100%,95%,70%, H2O) 1X5 min
3.Incubate the slides in EDTA for 50 min at 95°C for antigen unmasking
4.Cool the slides for 20 min at room temperature (RT), then wash once in H2O
7.Preincubate the slides with Ab mix for 20 min at RT
8.Incubate the slides ON at 4˚C with anti-γH2AX diluted in Ab mix at indicated concentration, then wash twice in TBS 1X
12.Dehydrate tissue sections through graded alcohol series and mount with Eukitt
13.Positive (irradiated skin) and negative (not irradiated skin) controls were included in each experiment
Approximately 5h
The specificity of the antibody in immunohistochemistry must be verified by testing known positive and negative controls. A positive control is the tissue in which the molecule of interest is known to be expressed and in the specific case a X-rays irradiated tissue. On the other side, the simplest negative control is the absence of expression in tissues in which the molecule of interest is known not to be expressed. A better negative control is the elimination of the signal by pre-incubating the antibody with an excess of the peptide or protein with which it was raised, or by replacing the primary antibody with buffer in Ab mix. Each single experiment should run positive as well as negative controls simultaneously with the testing samples.
Antigen unmasking can be optimized for different antibodies.
DAB solution must be prepared just before the use according to manufacturer’s protocol.
When tissue staining has not given the expected results, several experimental variables should be considered:
NO STAINING
1) Confirm that no reagents were omitted and that were added in the correct order and for sufficient incubation times.
2) Check antibody titrations and dilutions. This is particularly important for the primary antibody.
3) Check reagent expiration dates and storage.
4) Check specimen storage.
5) Check that the microscope is adjusted correctly.
6) Do not let the slides air dry in any step of the procedure
BACKGROUND
Immunocytochemical Methods and Protocols (second edition), edited by Lorette C. Javois, from Methods in Molecular Medicine, volume 115, Humana Press, 1999
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