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ATM / Serine-protein kinase ATM · IHC design guide
Design Immunohistochemistry for ATM
Use this guide to plan ATM staining in paraffin sections around its general nuclear tissue profile (HPA tissue IHC). Interpret goblet cell staining cautiously because HPA disregarded that signal (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ATM Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Predominantly nuclear (HPA tissue IHC; UniProt) | |
| Staining pattern | General nuclear staining across cell types (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | None in HPA (detected in all 44 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Goblet cell staining was disregarded (HPA tissue IHC) | |
| Regulation | Damage induces ATM autophosphorylation (UniProt) | |
| Isoform / epitope | No isoforms listed; chain spans residues 2–3056 (UniProt) |
Section 1
Recommended ATM IHC & IF Protocols
The catalog antibody’s IHC-P protocol is followed by three published protocols for ATM staining in paraffin sections, including one that measures phosphorylated ATM.
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A00014); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-ATM, 2.5 μg/mL (datasheet A00014) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ATM-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with the catalog antibody’s Tris-EDTA pH 9.0 retrieval at 95–98 °C for 20 min; the published retrieval conditions apply to their respective antibodies.
Section 2
What Is the Expected ATM Staining Pattern?
ATM is primarily nuclear and has no transmembrane segment (UniProt Q13315: localization, topology). In paraffin sections, expect a general nuclear pattern, including high staining reported in adrenal glandular cells, bronchial respiratory epithelial cells and bone marrow hematopoietic cells (HPA: tissue IHC). HPA rates the tissue profile Supported, with medium consistency between antibody staining and RNA expression; its reported goblet cell staining was disregarded (HPA: tissue IHC reliability).
| Distinct nuclear staining in glandular, respiratory epithelial or hematopoietic cells. | This fits ATM's primary nuclear localization and HPA's high staining calls for adrenal glandular, bronchial epithelial and bone marrow hematopoietic cells (UniProt Q13315: localization; HPA: tissue IHC). Judge localization within the identified cells, alongside a negative detection control (standard IHC practice). |
| Predominantly membrane-bound staining, with little or no nuclear signal. | A membrane-dominant pattern conflicts with ATM's primary nuclear location and lack of a transmembrane segment, so assess it as possible nonspecific staining (UniProt Q13315: localization, topology). Cytoplasmic signal alone is less decisive: UniProt also records vesicle, centrosome and peroxisome locations (UniProt Q13315: localization). |
| Strong staining in an unexpected cell population, especially if nuclei are unstained. | Consider cross-reactivity or endogenous chromogenic detection activity and check a negative detection control (standard IHC practice). ATM is broadly distributed, so an unlisted cell type is not automatically wrong; HPA reports low tissue specificity and provides no negative tissue call here (HPA: tissue IHC). |
| Diffuse color across tissue, extracellular spaces or slide background. | Poorly localized color cannot establish ATM expression (standard IHC practice). Compare with a negative detection control, then review blocking, washes and detection conditions (standard IHC practice); do not score diffuse background as HPA's general nuclear pattern (HPA: tissue IHC). |
| No nuclear signal in a section selected from an HPA high-staining tissue. | First check cell identity, tissue preservation and run controls, antibody application, detection and counterstain (standard IHC practice). Absence in one section does not establish biological loss: HPA's tissue profile has medium antibody–RNA consistency, and its high call applies to specified cells rather than every cell in that tissue (HPA: tissue IHC). |
💡Expected ATM appearanceCall the stain positive when identified HPA high-staining cell populations show clear, predominantly nuclear chromogenic signal; isolated membrane-dominant or diffuse color without corresponding nuclear staining is suspect (HPA: tissue IHC; UniProt Q13315: localization, topology; standard IHC practice).
| Tissue and cell selection | HPA reports high staining in adrenal glandular cells, bronchial respiratory epithelial cells and bone marrow hematopoietic cells, while caudate glial cells and ovarian stroma cells are listed as low (HPA: tissue IHC). Select and score the named cells; these calls do not define a universal negative control (HPA: tissue IHC). |
| Goblet cell signal | HPA lists high goblet cell staining in appendix, colon and duodenum but explicitly says goblet cell staining was observed and disregarded (HPA: tissue IHC). Do not use goblet cell color alone to validate an ATM positive result (HPA: tissue IHC reliability). |
| Antibody evidence | CAB000102 and CAB080442 have Supported IHC status; HPA067142 has no IHC status in the supplied antibody record (HPA: antibodies). An ICC Enhanced designation for HPA067142 does not establish its performance in paraffin IHC (HPA: antibodies). |
| Subcellular exceptions | UniProt describes ATM as primarily nuclear, with vesicle, centrosome and ROS-associated peroxisome localization also recorded (UniProt Q13315: localization). These exceptions warrant context when assessing cytoplasmic staining; they do not make a diffuse chromogenic haze diagnostic of ATM (UniProt Q13315: localization; standard IHC practice). |
| IF/ICC Q&A: where should ATM appear? | Mainly in nucleoplasm; HPA also reports vesicles and Golgi localization as uncertain (HPA: subcellular ICC-IF). This IF observation is context for localization, not an IHC staining or antibody-validation claim (HPA: subcellular ICC-IF; HPA: antibodies). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected high-staining cell population has no nuclear signal. | The selected cells may be absent from the section, or the staining run may have failed (HPA: tissue IHC; standard IHC practice). | Confirm cell identity, then check the positive run control, antibody application and chromogenic detection (standard IHC practice). Use HPA's high call as a reference for its specified cells, not for every cell in the tissue (HPA: tissue IHC). |
| Signal is predominantly at cell borders or membranes. | This conflicts with primary nuclear ATM localization and its lack of a transmembrane segment (UniProt Q13315: localization, topology). | Check whether nuclei stain in the same cells; compare with a negative detection control and reassess antibody specificity before scoring membrane color (standard IHC practice). |
| Color appears broadly in an unexpected cell type. | Cross-reactivity or endogenous detection activity is possible (standard IHC practice), but HPA reports low tissue specificity and supplies no negative tissue category here (HPA: tissue IHC). | Identify the stained cells, compare their compartment with the expected nuclear pattern, and inspect a negative detection control (HPA: tissue IHC; standard IHC practice). Avoid calling the cell type ATM-negative from this record alone (HPA: tissue IHC). |
| Goblet cells show strong color but nearby nuclei do not. | HPA explicitly disregarded the observed goblet cell staining, despite listing high levels in appendix, colon and duodenum (HPA: tissue IHC). | Do not treat goblet cell color as confirmation; evaluate nuclear staining in identified cells and check detection controls (HPA: tissue IHC; standard IHC practice). |
| The whole section has weak, diffuse chromogenic color. | Background from blocking, washing or detection can obscure cell boundaries (standard IHC practice). Such color does not resolve HPA's general nuclear pattern (HPA: tissue IHC). | Review blocking, wash steps and detection conditions, then compare with a negative detection control before assigning a cell-level score (standard IHC practice). |
| Nuclear staining varies between sections or antibody choices. | HPA reports only medium consistency between antibody staining and RNA expression; the supplied antibody records also have different application statuses (HPA: tissue IHC reliability; HPA: antibodies). | Check that the antibody has IHC support, compare the same identified cell population across sections, and interpret discordance with tissue controls (HPA: antibodies; standard IHC practice). Target-specific fixation sensitivity is unreported in the supplied sources (HPA: tissue IHC; UniProt Q13315). |
Experiment evidence
IHC & IF images showing ATM staining
A00014 · IHCImmunohistochemistry of ATM in human lymph node tissue with ATM antibody at 2.5 μg/mL.
A00014 · IFImmunofluorescence of ATM in Human Lymph Node cells with ATM antibody at 10 μg/mL.
M00014 · IHCImmunohistochemical analysis of paraffin-embedded human breast cancer, using ATM Antibody.
M00014 · IFImmunofluorescent analysis of Hela cells, using ATM Antibody.
P00014-1 · IHCImmunohistochemical analysis of paraffin-embedded human liver, using Phospho-ATM (S1981) Antibody.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000149311-ATM/tissue/cerebral+cortex#imid_409442
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000149311-ATM/tissue/colon#imid_409368
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000149311-ATM/tissue/liver#imid_409377
HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000149311-ATM/tissue/pancreas#imid_409320
HPA · ICC-IF ASC52teloHuman Protein Atlas immunofluorescence, ASC52telo, antibody HPA067142; green = ATM, blue = nucleus, red = microtubules.
HPA · ICC-IF ASC52teloHuman Protein Atlas immunofluorescence, ASC52telo, antibody HPA067142; green = ATM, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ATM IHC & IF
🧪Run bone marrow first: hematopoietic cells should stain (HPA: High in bone marrow hematopoietic cells), with predominantly nuclear signal (UniProt Q13315: primarily nuclear). HPA detects ATM in all 44 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and expect any unstained cells within the positive section to show counterstain only, without specific chromogen (HPA: no negative tissue; standard IHC practice).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: ATM is detected in all 44 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ATM in ASC52telo, EFO-21, U2OS, KOLF2.1J, HeLa BAC 2511, HeLa , with annotated localisation: Nucleoplasm (enhanced), Vesicles (uncertain) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, an isotype control matched to the primary antibody’s host species and immunoglobulin class, and ATM-knockout material as a biological negative if available (standard IHC practice). For bone marrow chromogenic IHC, quench endogenous peroxidase and check the control slide for residual staining in hematopoietic cells (HPA: bone marrow hematopoietic cells; standard IHC practice).
⚠️Feasibility: The supplied evidence reports no ATM-specific fixation window or antigen retrieval dependence, so retrieval requires empirical optimization (supplied evidence; standard IHC practice). The A00014 lymph-node IHC caption reports 2.5 µg/mL but leaves the fixative unreported (A00014 caption); the supplied evidence does not establish whether frozen sections or IF would be easier. In bone marrow, endogenous peroxidase can complicate chromogenic scoring, making the no-primary control useful for judging background (HPA: bone marrow hematopoietic cells; standard IHC practice).
HPA tissue IHC evidence for ATM
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — Medium consistency between antibody staining and RNA expression data. Goblet cell staining observed and disregarded.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Goblet cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: ATM is detected in all 44 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced ATM IHC Tips
Troubleshoot ATM staining in paraffin sections by checking retrieval, nuclear localisation, controls and cell-based scoring before interpreting chromogenic signal.
Which retrieval conditions should I try first for weak ATM staining?
Start with Tris-EDTA at pH 9.0, heated to 95–98 °C for 20 min (page protocol: nuclear antigen rule). If nuclear staining is weak, compare a shorter heating time on serial sections while holding antibody concentration and detection conditions constant (standard IHC practice). If that does not help, test citrate at pH 6.0 as a fallback, with a matched control section in the same run (standard IHC practice). Score recovery in intact nuclei because ATM is primarily nuclear, while excessive retrieval can damage section morphology and complicate scoring (UniProt Q13315 localisation; standard IHC practice).
Could fixation explain weak ATM staining in my paraffin sections?
ATM-specific sensitivity to fixation is unknown from the supplied evidence; the selected tissue caption does not state a fixative (selected IHC caption: A00014). Record the actual fixative and fixation duration for each specimen, then compare sections processed with the same retrieval and detection conditions (standard IHC practice). Check morphology and nuclear counterstain before increasing antibody concentration, since damaged or poorly preserved nuclei make nuclear scoring unreliable (standard IHC practice; UniProt Q13315 localisation). If fixation histories differ, treat a staining difference as inconclusive until matched material or a controlled processing comparison separates specimen handling from ATM expression (standard IHC practice).
How should I assess cytoplasmic ATM staining alongside nuclear signal?
Expect the main ATM signal in nuclei, consistent with its primarily nuclear location and the reported general nuclear tissue expression (UniProt Q13315 localisation; HPA tissue IHC profile). ATM also has reported cytoplasmic vesicle, centrosome and peroxisome locations, so discrete nonnuclear signal needs context rather than automatic rejection (UniProt Q13315 localisation). Compare its distribution with intact nuclear staining, section morphology and a negative control under identical chromogenic conditions (standard IHC practice). Score nuclear and nonnuclear staining separately, and avoid calling diffuse cytoplasmic colour a compartment-specific ATM signal without independent validation (standard IHC practice).
How can I troubleshoot discordant results from two ATM antibodies?
First check each antibody’s documented immunogen or epitope and whether it recognizes total or modified ATM; those details are not supplied for A00014 here (selected IHC caption: A00014). The supplied record lists 0 isoforms and several phosphorylation sites, including Ser1981, so do not explain disagreement by an unlisted isoform or assume both reagents detect the same phosphorylation state (UniProt Q13315 isoforms and modified residues). Run serial sections with matched retrieval and appropriate negative controls, keeping each antibody’s detection conditions documented (standard IHC practice). Compare nuclear localisation and tissue distribution before interpreting discordance as biology (UniProt Q13315 localisation; standard IHC practice).
How should I adapt ATM localisation checks to multiplex immunofluorescence?
Use a marker for the expected cell population, such as a validated hematopoietic-cell marker when examining bone marrow, and pair it with a nuclear counterstain (HPA tissue IHC: high in bone marrow hematopoietic cells; standard IF practice). Choose fluorophores after viewing unstained tissue, assigning the ATM channel away from strong tissue autofluorescence where possible (standard IF practice). Because ATM has no transmembrane segment and is primarily nuclear, test controlled permeabilisation that gives antibody access to intracellular and nuclear epitopes while preserving cell structure (UniProt Q13315 topology and localisation; standard IF practice). Include single-channel and secondary-only controls before interpreting vesicular signal, which has uncertain subcellular support (HPA subcellular: vesicles uncertain; standard IF practice).
What should I check when ATM DAB staining is widespread?
Examine a section without primary antibody to assess detection-system background, and include a peroxidase block before DAB development (standard chromogenic IHC practice). Titrate the primary antibody and shorten DAB development if colour obscures nuclei; the selected A00014 caption reports 2.5 µg/mL in human lymph node tissue, without establishing an optimal concentration for every specimen (selected IHC caption: A00014; standard IHC practice). Review section edges, folds and damaged regions separately from well-preserved tissue (standard IHC practice). General nuclear expression is reported, but diffuse chromogen outside recognisable cells should not be scored as ATM (HPA tissue IHC profile; standard IHC practice).
How should I quantify ATM staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the compartment before scoring: use nuclear intensity and the percentage of positive nuclei, then calculate an H-score if graded intensity is reproducible (UniProt Q13315 localisation; standard IHC practice). Count within a specified cell population and tissue area, normalising positive nuclei to all evaluable nuclei of that population rather than to total section area (standard IHC practice). For sparse populations, report positive-cell density per mm² alongside the denominator and excluded regions (standard IHC practice). Keep retrieval, antibody concentration, DAB development, counterstain and image thresholds constant, and report nonnuclear staining separately because ATM has additional reported locations (standard IHC practice; UniProt Q13315 localisation).
When is an apparent ATM-positive cell likely to be an artefact?
A convincing result shows signal in intact nuclei within identifiable cells, consistent with ATM’s primarily nuclear localisation and the general nuclear tissue profile (UniProt Q13315 localisation; HPA tissue IHC profile). Treat staining confined to cut edges, folds or necrotic areas as suspect, and check it against the no-primary control for detection background (standard IHC practice). Investigate granular cytoplasmic signal separately because vesicular ATM is reported, but its HPA vesicle assignment is uncertain (UniProt Q13315 localisation; HPA subcellular: vesicles uncertain). Cell identity also matters: HPA explicitly disregarded goblet-cell staining, so a strong goblet-cell signal alone is weak evidence of specific ATM detection (HPA tissue IHC reliability description).
Boster reagents
Best ATM / Serine-protein kinase ATM IHC Antibodies
Human-reactive ATM antibodies have IHC images from lymph node, breast cancer, and liver (catalog IHC image captions); IF images show human lymph node cells and HeLa cells (catalog IF image captions).
Real IHC data
Anti-Serine-protein kinase ATM ATM Antibody
Cat # A00014
Real IHC data
Anti-ATM Monoclonal Antibody
Cat # M00014
Real IHC data
Anti-Phospho-ATM (S1981) Rabbit Monoclonal Antibody
Cat # P00014-1
A00014 has pictured IHC in human lymph node and IF in human lymph node cells; M00014 has pictured IHC in paraffin-embedded human breast cancer and IF in HeLa cells (catalog image captions). P00014-1 has pictured IHC in paraffin-embedded human liver and is listed for IF without an IF image (catalog image caption and applications).
Which to pick: For tissue IHC, choose A00014 for human lymph node (A00014 IHC image caption), M00014 for paraffin-embedded human breast cancer (M00014 IHC image caption), or P00014-1 when detecting phospho-ATM S1981 in paraffin-embedded human liver (P00014-1 title and IHC image caption); the captions do not report a fixative. For IF/ICC, M00014 is a monoclonal antibody listed for both applications, with pictured IF in HeLa cells (catalog clone, applications, and IF image caption); A00014 is listed for IF and has pictured IF in human lymph node cells (catalog applications and IF image caption). For human, mouse, or rat IF/ICC, consider monoclonal M00014-1; it is listed for those species and applications but has no IHC application or IHC image (catalog reactivity, clone, applications, and image list).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q13315 (ATM_HUMAN, Serine-protein kinase ATM).
- Human Protein Atlas. ATM tissue IHC expression (reliability: Supported).
- Human Protein Atlas. ATM subcellular location (ICC-IF): Mainly localized to the nucleoplasm and vesicles. In addition localized to the Golgi apparatus..
- Human Protein Atlas. ATM antibody validation summary (3 antibodies).
- ATM Germline-Mutated Gastroesophageal Junction Adenocarcinomas: Clinical Descriptors, Molecular Characteristics, and Potential Therapeutic Implications. Journal of the National Cancer Institute 2022 — PMC9086803.
- Genomic and Clinicopathologic Characterization of ATM-deficient Prostate Cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 2020 — PMC7501149.
- Having pancreatic cancer with tumoral loss of ATM and normal TP53 protein expression is associated with a poorer prognosis. Clinical cancer research : an official journal of the American Association for Cancer Research 2014 — PMC3975663.
- Impact of SAMHD1 and phosphorylated ATM levels on the progression and prognosis of patients with soft tissue sarcoma. Oncology letters 2025 — PMC12512546.
- PubMed PMID:8589678 — UniProt-cited evidence.
- PubMed PMID:8665503 — UniProt-cited evidence.
- PubMed PMID:9199932 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ATM (Serine-protein kinase ATM). Expected localisation Predominantly nuclear (HPA tissue IHC; UniProt). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A00014 at 2.5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative None in HPA (detected in all 44 tissues); use no-primary + isotype controls. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet A00014); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-ATM, 2.5 μg/mL (datasheet A00014); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Goblet cell staining was disregarded (HPA tissue IHC); Isoform / epitope: No isoforms listed; chain spans residues 2–3056 (UniProt); Regulation: Damage induces ATM autophosphorylation (UniProt). Evidence: UniProt Q13315, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the nucleoplasm and vesicles. In addition…, Datasheet A00014, PMC7501149, PMC3975663. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ATM/immunohistochemistry-ihc-guide.