ATM / Serine-protein kinase ATM · IHC design guide

Design Immunohistochemistry for ATM

Plan chromogenic ATM IHC in paraffin sections around the general nuclear tissue pattern (HPA tissue IHC). A human lymph node IHC example uses catalog antibody A00014 at 2.5 μg/mL (catalog IHC caption; datasheet).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for ATM (IHC for ATM): expected localisation General nuclear tissue staining (HPA tissue IHC); primarily nuclear (UniProt), antibody A00014, validated IHC image, and IHC protocol steps — Printable **ATM** IHC protocol sheet — expected localisation General nuclear tissue staining (HPA tissue IHC); primarily nuclear (UniProt), antibody A00014, controls and protocol steps. Open the full ATM IHC guide →

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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	General nuclear tissue staining (HPA tissue IHC); primarily nuclear (UniProt)
	Staining pattern	Nuclear staining in glandular and hematopoietic cells (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 observed and disregarded (HPA tissue IHC)
	Regulation	Low tissue specificity at the RNA level (HPA tissue RNA)
	Isoform / epitope	No isoforms reported; mature chain spans residues 2–3056 (UniProt)

Section 1

Recommended ATM IHC & IF Protocols

Compare the catalog antibody’s IHC-P protocol with published ATM IHC in prostate and pancreatic tumors, plus a distinct phospho-ATM assay in sarcoma (PMC7501149; PMC3975663; PMC12512546).

Recommended immunohistochemistry (IHC-P) protocol parameters
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 Tris-EDTA pH 9.0 retrieval for the catalog antibody (page retrieval specification); evaluate published retrieval conditions when adapting a cited assay.

Section 2

What Is the Expected ATM Staining Pattern?

ATM is primarily nuclear (UniProt Q13315 subcellular location) and has no transmembrane segment (UniProt Q13315 topology). In paraffin section IHC, expect nuclear staining across several cell populations, including adrenal and breast glandular cells, bronchial respiratory epithelial cells, and bone marrow hematopoietic cells (HPA tissue IHC: High). HPA describes general nuclear expression with Supported reliability; it reports only medium consistency between staining and RNA expression (HPA tissue IHC).

What am I looking at on my slide?
Distinct nuclear staining in adrenal or breast glandular cells, bronchial respiratory epithelium, or bone marrow hematopoietic cells.	This fits ATM’s primarily nuclear location (UniProt Q13315 subcellular location) and the High staining reported for those cells (HPA tissue IHC). Compare cells within the same section before judging intensity: HPA reports low tissue RNA specificity, so a single exclusive positive population is not expected (HPA tissue IHC: Low tissue specificity).
Vesicular or other cytoplasmic signal appears alongside clear nuclear staining.	A secondary compartment can be plausible: UniProt lists cytoplasmic vesicles and peroxisomes, and HPA ICC-IF reports enhanced nucleoplasmic localization with uncertain vesicular localization (UniProt Q13315 subcellular location; HPA subcellular ICC-IF). Interpret the nuclear pattern first; vesicle-like chromogenic deposits alone provide weaker evidence of specific ATM staining.
Strong signal is confined to a compartment other than nuclei, with little nuclear staining.	Treat this as a discordant IHC pattern because UniProt calls ATM primarily nuclear and HPA describes general nuclear tissue expression (UniProt Q13315 subcellular location; HPA tissue IHC). Check whether the signal follows tissue pigment, precipitate, or an endogenous detection reaction before scoring it as ATM (general IHC practice).
Goblet cells dominate the positive field, or staining appears only in a cell population absent from the expected pattern.	HPA explicitly observed and disregarded goblet cell staining despite listing High staining in appendix, colon, and duodenum goblet cells (HPA tissue IHC). Do not use that signal alone to confirm specificity. An isolated unexpected cell pattern can reflect antibody cross-reactivity or endogenous detection activity; assess controls and nuclear localization (general IHC practice).
A known-positive tissue shows diffuse background or no interpretable nuclear signal.	Diffuse staining cannot establish the expected nuclear pattern (HPA tissue IHC: General nuclear expression). An absent signal in a HPA High population, such as bone marrow hematopoietic cells, is a failed positive-control result rather than evidence that the specimen lacks ATM (HPA tissue IHC: High in hematopoietic cells; general IHC practice).

💡Expected ATM appearanceCall a section positive when identifiable nuclei show distinct chromogenic staining in an HPA High population such as adrenal glandular or bone marrow hematopoietic cells (HPA tissue IHC), consistent with ATM’s primary nuclear location (UniProt Q13315); isolated goblet cell staining or dominant extranuclear deposit is not a convincing positive pattern (HPA tissue IHC: goblet staining disregarded; general IHC practice).

How each factor affects the staining
Tissue and cell selection	Use an HPA High nuclear-staining population as a positive comparator, such as adrenal or breast glandular cells, bronchial respiratory epithelial cells, or bone marrow hematopoietic cells (HPA tissue IHC). Glial cells in caudate and ovarian stroma are listed Low; weak staining there is a poor standalone failure test (HPA tissue IHC: Low).
Evidence strength and antibody choice	The tissue pattern has Supported reliability and medium staining–RNA consistency (HPA tissue IHC). CAB000102 and CAB080442 have Supported IHC validation, whereas HPA067142 has Enhanced ICC validation without an IHC status in the supplied record (HPA antibodies). Match the antibody’s validation to the readout being interpreted.
Subcellular readout: IF/ICC Q&A	Q: Should IF/ICC show only nuclei? A: HPA reports enhanced nucleoplasm, uncertain vesicles, and uncertain additional Golgi localization (HPA subcellular ICC-IF). UniProt also lists cytoplasmic vesicles and ROS-dependent peroxisomal translocation (UniProt Q13315 subcellular location). These observations inform interpretation, but IF/ICC requires its own guide.
Target structure and staining boundary	ATM has no transmembrane segment, signal peptide, or propeptide, and its recorded chain spans residues 2–3056 (UniProt Q13315 topology and processing). A membrane outline or luminal surface pattern alone is therefore discordant with the supplied target annotation; the record does not establish a tissue IHC fixation effect.
Activation state	DNA damage can trigger ATM autophosphorylation and active monomers (UniProt Q13315 subunit and function). The supplied HPA tissue profile describes general nuclear expression, not a damage-state readout (HPA tissue IHC). Do not infer ATM activation from total-ATM chromogenic intensity without a separately validated activation-specific assay (general IHC practice).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
No nuclear stain in the positive-control section.	Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation.	Repeat with a documented HPA High population, confirm the catalog antibody’s IHC-P conditions, and check retrieval, detection, and counterstain using the assay controls (HPA tissue IHC; general IHC practice).
Only goblet cells stain strongly.	HPA observed goblet cell staining and explicitly disregarded it, including in tissues where goblet cells are listed High (HPA tissue IHC).	Score another HPA High cell population with nuclear morphology and compare an appropriate antibody or detection control before assigning ATM positivity (HPA tissue IHC; general IHC practice).
Brown reaction product fills cells or tissue spaces and obscures nuclei.	Diffuse background or endogenous detection activity can hide the expected nuclear pattern (HPA tissue IHC: General nuclear expression; general IHC practice).	Inspect a no-primary control, review blocking and wash steps, and adjust detection exposure according to the assay instructions; then reassess nuclear contrast (general IHC practice).
Staining is mainly at membranes or in coarse extracellular deposits.	That distribution conflicts with ATM’s primarily nuclear location and lack of a transmembrane segment (UniProt Q13315 subcellular location and topology).	Check for pigment or precipitate and compare the no-primary control; score only a reproducible nuclear component in appropriate cells (general IHC practice; HPA tissue IHC).
A weak caudate or ovarian-stroma section appears negative.	HPA lists caudate glial cells and ovarian stroma cells as Low, so they offer limited sensitivity as sole positive controls (HPA tissue IHC).	Run an HPA High comparator in parallel before calling the assay negative; evaluate comparable cell types and nuclear signal rather than whole-section color (HPA tissue IHC; general IHC practice).
IF/ICC shows puncta, but tissue IHC gives little nuclear staining.	HPA marks IF/ICC vesicles uncertain and its enhanced ICC antibody has no supplied IHC status; the two readouts have different validation records (HPA subcellular ICC-IF; HPA antibodies).	Judge paraffin IHC against the antibody’s IHC validation and HPA nuclear tissue profile. Use the separate IF/ICC guide to interpret puncta; do not treat them as a substitute for an IHC positive control (HPA antibodies; HPA tissue IHC).

Source: UniProt Q13315 Human Protein Atlas tissue Human Protein Atlas subcellular

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.

← 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). HPA detects ATM in all 44 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and expect mature erythrocytes on the positive slide to lack nuclear ATM staining because they have no nuclei (standard histology).

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 slide and an isotype control matched to the primary antibody’s host species, clonality and concentration; an ATM knockout specimen, if available, provides a biological specificity control (standard IHC practice). In bone marrow, quench endogenous peroxidase and check for residual chromogen signal in blood cells (standard IHC practice).

⚠️Feasibility: A target-specific fixation window and retrieval dependency are unreported in the supplied evidence; optimize antigen retrieval for the catalog antibody on paraffin sections (standard IHC practice). The selected A00014 tissue-IHC caption reports 2.5 μg/mL in human lymph node but leaves the fixative unreported (A00014 caption). The evidence does not establish whether frozen sections or IF are easier for ATM; bone marrow blood cells can produce endogenous peroxidase artefact in chromogenic IHC (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 nuclear localisation, tissue controls and scoring consistency before interpreting signal changes (UniProt Q13315; HPA tissue IHC).

How should I retrieve ATM antigen when nuclear staining is weak?

Start with Tris-EDTA pH 9.0 heat-induced retrieval at 95–98 °C for 20 min (page retrieval: nuclear antigen). Keep section thickness, heating and cooling consistent across control and test slides so differences in nuclear signal remain interpretable (standard IHC practice). If staining is weak, compare a shorter retrieval exposure on adjacent sections while holding antibody concentration and detection conditions constant (standard IHC practice). Check for nuclear staining in a concurrently processed positive control, since ATM is primarily nuclear and the HPA tissue profile reports general nuclear expression (UniProt Q13315 localisation; HPA tissue IHC).

Could fixation explain absent or uneven ATM staining?

ATM-specific fixation sensitivity is unknown from the supplied evidence; the selected tissue image caption does not state its fixative (A00014 tissue-IHC caption). Record the specimen fixative, fixation duration and processing history before comparing sections, because those conditions can change antigen accessibility in paraffin IHC (standard IHC practice). Run differently processed specimens through the same retrieval, antibody incubation and chromogenic detection workflow, with a matched positive control in each batch (standard IHC practice). If signal remains uneven, examine section folds, drying and tissue preservation before assigning the pattern to ATM biology (standard IHC practice). Do not infer fixation tolerance from ATM localisation or tissue staining patterns (UniProt Q13315 localisation; HPA tissue IHC).

Which ATM staining compartment should I accept in tissue sections?

Expect predominantly nuclear staining in interpretable cells, consistent with ATM's primary nuclear localisation and the HPA general nuclear tissue profile (UniProt Q13315 localisation; HPA tissue IHC). Compare chromogenic signal with the nuclear counterstain at high magnification, then score nuclear and cytoplasmic compartments separately (standard IHC practice). ATM is also reported in cytoplasmic vesicles, centrosomes and the peroxisome matrix, so a reproducible extranuclear pattern merits compartment-specific validation (UniProt Q13315 localisation). HPA subcellular data place ATM mainly in nucleoplasm and report vesicle and Golgi assignments as uncertain; do not treat those uncertain assignments as proof of tissue staining specificity (HPA subcellular).

How do ATM domains and modifications affect epitope troubleshooting?

Check the catalog antibody's stated immunogen or epitope before attributing staining changes to a particular ATM region (standard IHC practice). The supplied record lists 0 isoforms and places the FAT, kinase and FATC domains at residues 1940–2566, 2686–2998 and 3024–3056, respectively (UniProt Q13315). ATM also has documented phosphorylation sites, including residues 367, 1893, 1981 and 1983 (UniProt Q13315). Unless the antibody's epitope and modification sensitivity are known, a change in chromogenic signal cannot distinguish altered protein abundance from altered epitope accessibility (standard IHC interpretation). Compare adjacent sections under identical retrieval and detection conditions before drawing that distinction (standard IHC practice).

How can IF help investigate an ambiguous ATM IHC pattern?

Use IF as a separate localisation check, pairing ATM with a marker for the expected cell type and a nuclear counterstain (standard IF practice). Select spectrally separated fluorophores and inspect unstained tissue for autofluorescence before assigning weak extranuclear signal to ATM (standard IF practice). ATM has no transmembrane segment and is primarily nuclear, so permeabilise fixed cells sufficiently to expose intracellular epitopes while preserving nuclear morphology (UniProt Q13315 topology and localisation; standard IF practice). Compare the resulting nuclear pattern with the chromogenic section, while treating HPA vesicle and Golgi assignments as uncertain rather than confirmed tissue localisation (HPA subcellular). Keep IF antibody validation separate from the IHC evidence for the selected tissue image (A00014 tissue-IHC caption; standard assay validation practice).

How do I reduce diffuse brown background without losing ATM signal?

Assess a section without primary antibody to identify background from detection reagents, tissue pigment or endogenous enzyme activity (standard chromogenic IHC practice). Include the usual peroxidase block before HRP-based DAB detection, and check whether brown signal persists in the no-primary control (standard chromogenic IHC practice). Titrate the primary antibody against a positive control; the selected ATM tissue image reports 2.5 µg/mL, but its caption does not establish a universal working concentration (A00014 tissue-IHC caption). Optimise blocking and washing one variable at a time, then confirm that predominantly nuclear staining remains visible (standard IHC practice; UniProt Q13315 localisation).

What is a defensible way to quantify ATM IHC? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and nuclear scoring threshold before reviewing outcome groups, because ATM is primarily nuclear and HPA describes general nuclear expression (UniProt Q13315 localisation; HPA tissue IHC). Report the percentage of positive nuclei and an H-score based on staining intensity, or positive nuclei per mm² when cell density is the relevant endpoint (standard IHC scoring practice). Normalise positive counts to the total number of eligible nuclei in the same annotated compartment, excluding folds, necrosis and damaged edges (standard IHC scoring practice). Keep retrieval, detection, counterstain and image analysis settings consistent across batches, and include the same reference tissue for batch comparisons (standard IHC practice).

When is apparent ATM positivity likely to be artefactual?

Give greatest weight to staining that follows intact nuclei in the expected cell population and recurs in independently processed sections (UniProt Q13315 localisation; standard IHC interpretation). Be cautious with isolated cytoplasmic brown deposits: ATM has reported extranuclear sites, but HPA vesicle and Golgi assignments are uncertain (UniProt Q13315 localisation; HPA subcellular). Exclude edge effects, necrotic areas and signal retained in a no-primary control before calling a sample positive (standard chromogenic IHC practice). HPA reports that goblet cell staining was observed and disregarded, so do not use that pattern alone as validation of ATM positivity (HPA tissue IHC). Check persistent brown signal against the peroxidase block and detection controls (standard chromogenic IHC practice).

Boster reagents

Best ATM / Serine-protein kinase ATM IHC Antibodies

The catalog shows human ATM IHC images from lymph node, breast cancer and liver, plus IF images from lymph node cells and HeLa cells (catalog image captions).

Real IHC data Immunohistochemistry of ATM in human lymph node tissue with ATM antibody at 2.5 μg/mL.

Anti-Serine-protein kinase ATM ATM Antibody

Cat # A00014

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Real IHC data Immunohistochemical analysis of paraffin-embedded human breast cancer, using ATM Antibody.

Anti-ATM Monoclonal Antibody

Cat # M00014

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Real IHC data Immunohistochemical analysis of paraffin-embedded human liver, using Phospho-ATM (S1981) Antibody.

Anti-Phospho-ATM (S1981) Rabbit Monoclonal Antibody

Cat # P00014-1

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A00014 has IHC and IF images from human lymph node tissue and cells, respectively; M00014 has IHC from paraffin-embedded human breast cancer and IF from HeLa cells (catalog image captions). P00014-1 has an IHC image from paraffin-embedded human liver and is listed for IF without an IF image (catalog image captions; catalog applications).

Which to pick: For tissue IHC, choose A00014 for human lymph node at 2.5 μg/mL, M00014 for paraffin-embedded human breast cancer, or P00014-1 when targeting phospho-ATM (S1981) in paraffin-embedded human liver (respective IHC captions; catalog titles); the fixative is unreported (catalog IHC captions). For IF/ICC, A00014 has IF imaging in human lymph node cells, while monoclonal M00014 has IF imaging in HeLa cells and lists ICC (respective IF captions; catalog clone and applications). For human, mouse or rat IF/ICC, consider monoclonal M00014-1 (catalog reactivity, clone and applications); it has no listed IHC application or IHC image (catalog applications and image captions).

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.