AR / Androgen receptor · IHC design guide

Design Immunohistochemistry for AR

Plan paraffin-section AR IHC around the nuclear staining seen mainly in reproductive organs (HPA tissue IHC). Use consistent fixation and account for ligand-dependent movement of AR into nuclei when interpreting staining (UniProt).

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

Immunohistochemistry protocol sheet for AR (IHC for AR): expected localisation Nuclear staining in tissues (HPA tissue IHC); cytoplasmic when unliganded (UniProt), antibody M00542-1, validated IHC image, and IHC protocol steps — Printable AR IHC protocol sheet — expected localisation Nuclear staining in tissues (HPA tissue IHC); cytoplasmic when unliganded (UniProt), antibody M00542-1, controls and protocol steps. Open the full AR IHC guide →

Navigate to: Protocols Staining pattern Controls Antibodies

AR 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	Nuclear staining in tissues (HPA tissue IHC); cytoplasmic when unliganded (UniProt)
	Staining pattern	Nuclear staining in several tissues, chiefly reproductive organs (HPA tissue IHC)
	Antigen retrieval	Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen)
	Positive control ⓘ	Epididymis+4 more · see all
	Negative control ⓘ	Adipose tissue+4 more · see all

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Keep fixation conditions consistent across paraffin sections. (standard IHC practice; not target-specific)
	Caveat	Ligand binding can shift AR staining into nuclei (UniProt)
	Regulation	Androgen promotes nuclear accumulation (UniProt)
	Isoform / epitope	Four isoforms; antibody epitope coverage is unspecified (UniProt)

Section 1

Recommended AR IHC & IF Protocols

The catalog antibody’s IHC-P protocol is accompanied by one published AR staining protocol for breast cancer biopsies (PMC13515587).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human prostate carcinoma tissue; fixative not specified (datasheet M00542-1)
Fixation	Image fixative and duration unreported (datasheet M00542-1); 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 monoclonal (clone HD-1) anti-AR, 1:50-1:100 (datasheet M00542-1)
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	AR-positive staining in glandular cells of epididymis (HPA tissue IHC: High). HPA tissue profile: Nuclear expression in several tissues, mainly in reproductive organs. No signal in the no-primary control.

💡Decision noteStart with Tris-EDTA pH 9.0 retrieval at 95–98 °C for 20 min (page retrieval rule for nuclear antigens).

Section 2

What Is the Expected AR Staining Pattern?

AR is a non-membrane receptor that occupies the cytoplasm and nucleus; ligand binding usually shifts it toward the nucleus (UniProt P10275 topology and subcellular location). In paraffin-section IHC, expect chiefly nuclear staining in selected cells, especially epididymal and seminal-vesicle glandular cells (HPA tissue IHC: Enhanced reliability; High staining in both). Interpret the compartment alongside the tissue, cell type, and assay used.

What am I looking at on my slide?
Distinct nuclear staining in epididymal or seminal-vesicle glandular cells, with little staining in adjacent unstained cells.	This matches the strongest supplied tissue examples (HPA tissue IHC: High in both glandular-cell populations). Nuclear localization also fits ligand-associated AR movement (UniProt P10275 subcellular location). Score the relevant cells and their nuclei; a positive area elsewhere on the section does not by itself validate the result.
A membrane-like outline or staining confined to unrelated structures, without convincing nuclear signal in expected positive cells.	A membrane pattern conflicts with AR's lack of a transmembrane segment (UniProt P10275 topology). Check the cellular boundary and counterstain before calling it AR. Cytoplasmic signal alone needs a more careful judgment: unligated AR can be cytoplasmic (UniProt P10275 subcellular location).
Strong staining in a cell population listed as undetected while the expected glandular or stromal cells are unstained.	Consider cross-reactivity or endogenous chromogen-generating activity before assigning AR expression. HPA reports, for example, no staining in adipocytes and adrenal glandular cells (HPA tissue IHC: Not detected). Those entries describe observed staining, not proof that every individual specimen must be negative.
A diffuse chromogenic haze across nuclei, cytoplasm, and extracellular space that obscures cell boundaries.	This cannot support a compartment-specific AR call. Reassess blocking, detection background, washing, and the no-primary control as general IHC checks. The expected tissue pattern is nuclear in several tissues, mainly reproductive organs (HPA tissue IHC: Enhanced reliability; profile).
No staining in epididymal or seminal-vesicle glandular cells, with an otherwise readable counterstain.	Treat the run as unresolved before concluding that AR is absent: both are High-staining examples (HPA tissue IHC). Check that the chosen tissue contains the named cell population, then review antibody validation, detection controls, and the assay's established retrieval conditions as general IHC troubleshooting.

💡Expected AR appearanceCall a positive result when glandular-cell nuclei show clear, cell-resolved chromogen in epididymis or seminal vesicle (HPA tissue IHC: High); a membrane rim or uniform section-wide haze is suspicious (UniProt P10275 topology; HPA tissue IHC: mainly nuclear profile).

How each factor affects the staining
Ligand state and compartment	AR is predominantly cytoplasmic without ligand and usually moves to the nucleus after ligand binding; unligated nuclear movement can also occur with RACK1 (UniProt P10275 subcellular location). A mixed nuclear–cytoplasmic pattern therefore needs biological context, while the HPA paraffin-tissue reference remains mainly nuclear (HPA tissue IHC profile).
Cell type and tissue selection	Epididymal and seminal-vesicle glandular cells stain High; breast and fallopian-tube glandular cells, endometrial stromal cells, renal proximal-tubule cell bodies, and cells in testicular seminiferous ducts stain Medium (HPA tissue IHC). Compare the same cell type across sections; a whole-tissue average can hide a valid cellular pattern.
Strength of the tissue reference	The HPA tissue profile is rated Enhanced because antibody staining is highly consistent with RNA expression data (HPA tissue IHC reliability). Two listed antibodies have Enhanced IHC status, CAB000001 and CAB065764 (HPA antibody validation). This supports the reported pattern; it does not validate an unlisted antibody or every staining condition.
Isoforms and unknown epitope coverage	UniProt lists four AR isoforms (UniProt P10275 isoforms). The supplied sources give no epitope position or isoform coverage for the catalog antibody, so an unexpected result cannot be assigned to a particular isoform from these data. Confirm the antibody's documented target region before interpreting isoform-specific staining.
Detection system background	Endogenous enzyme activity or nonspecific detection-reagent binding can create chromogen independently of the primary antibody (general IHC practice). Use an appropriate no-primary control and the detection system's blocking steps to distinguish that background from cell-resolved nuclear staining (HPA tissue IHC: mainly nuclear profile). This is a general workflow consideration, not an AR-specific effect.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Expected positive glandular cells are blank.	The section may miss the relevant cells, or a staining or detection step may have failed; the named glandular cells are High in the reference (HPA tissue IHC).	Verify cell identity on the counterstain, then check a known-positive section and detection controls. Review the validated antibody's documented IHC conditions, including retrieval, without assuming an AR-specific retrieval requirement.
Only a strong membrane rim appears.	That compartment is inconsistent with AR's lack of a transmembrane segment (UniProt P10275 topology); nonspecific staining is a possibility.	Check the rim against the counterstain and a no-primary control. Reassess antibody dilution and detection background using the assay's validated conditions before scoring the tissue.
Expected nuclei are obscured by diffuse brown background.	Blocking, washing, or detection background may prevent a cell-resolved readout (general IHC practice).	Compare with a no-primary control and inspect the detection and wash steps. Repeat under validated conditions, then score only identifiable cells and nuclei; the tissue reference is mainly nuclear (HPA tissue IHC profile).
Unexpected cell types stain strongly.	Cross-reactivity or endogenous detection activity is possible, particularly when a population differs from an HPA Not detected entry (HPA tissue IHC).	Identify the stained cell type, compare it with the relevant HPA entry, and examine the no-primary control. Do not infer an AR-positive cell solely from chromogen in a different population.
Signal is cytoplasmic as well as nuclear in an IHC-positive tissue.	Cytoplasmic AR is biologically plausible without ligand, although the HPA tissue-IHC profile emphasizes nuclei (UniProt P10275 subcellular location; HPA tissue IHC profile).	Record nuclear and cytoplasmic staining separately. Confirm cell identity and compare with a known-positive section before treating cytoplasmic signal alone as evidence of a failed or successful run.
IF/ICC Q&A: Is cytosolic AR signal expected?	Yes: HPA reports a supported cytosolic location in ICC-IF, and UniProt describes predominantly cytoplasmic unligated AR (HPA subcellular ICC-IF; UniProt P10275 subcellular location).	Interpret the IF/ICC compartment in its own assay context and consult the separate IF/ICC guide for its workflow. Do not use the paraffin-section nuclear IHC pattern as an exclusive IF/ICC acceptance rule.

Source: UniProt P10275 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing AR staining

M00542-1 · IHCImmunohistochemical analysis of paraffin-embedded human prostate carcinoma, using Androgen Receptor Antibody.

M00542-1 · IFImmunofluorescent analysis using the Antibody at 1:50 dilution.

M00542-2 · IHCImmunohistochemical analysis of paraffin-embedded human prostate, using Androgen Receptor Antibody.

M00542-2 · IFImmunofluorescent analysis using the Antibody at 1:50 dilution.

M00542-3 · IHCImmunohistochemical analysis of paraffin-embedded human prostate carcinoma, using Androgen Receptor(AR-V7 specific) Antibody.

IF analysis of Androgen Receptor/AR using anti-Androgen Receptor/AR antibody (A00542). Androgen Receptor/AR was detected in immunocytochemical section of T47D cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5μg/mL rabbit anti-Androgen Receptor/AR Antibody (A00542) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

A00542 · IFIF analysis of Androgen Receptor/AR using anti-Androgen Receptor/AR antibody (A00542). Androgen Receptor/AR was detected in immunocytochemic

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000169083-AR/tissue/cerebral+cortex#imid_57270

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000169083-AR/tissue/colon#imid_57196

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000169083-AR/tissue/liver#imid_57205

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000169083-AR/tissue/kidney#imid_57214

HPA · ICC-IF HAP1Human Protein Atlas immunofluorescence, HAP1, antibody HPA004733; green = AR, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for AR IHC & IF

🧪Run epididymis first: glandular cells should stain (HPA: High in epididymis glandular cells). Use adipose tissue as the negative: adipocytes should lack detectable staining (HPA: Not detected in adipocytes); on the positive slide, assess adjacent non-glandular cells as internal negatives only if they show no specific signal, because their AR status is not specified in the supplied HPA row.

Positive control tissue: Epididymis (Glandular cells, HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show AR in HAP1, Rh30, U2OS, with annotated localisation: Cytosol (supported) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species and clonality; use AR-knockout material as a biological negative where available (standard IHC practice). Block endogenous peroxidase for chromogenic detection, and check the epididymis slide for nonspecific signal in glandular lumens (standard IHC practice).

⚠️Feasibility: A target-specific fixation window, fixation effect, and retrieval dependency are unreported in the supplied evidence; optimize antigen retrieval for the catalog antibody (standard IHC practice). The selected M00542-1 paraffin-section caption does not report a fixative (selected product caption: fixative not stated), and the evidence does not establish whether frozen sections or IF are easier. In glandular tissue, avoid scoring luminal deposit as cellular staining; assess nuclear and cytoplasmic signal separately because AR can move from cytoplasm to nucleus after ligand binding (UniProt P10275: subcellular location).

HPA tissue IHC evidence for AR

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High consistency between antibody staining and RNA expression data.). 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
Epididymis	Glandular cells	High	Protein (IHC)	HPA →
Seminal vesicle	Glandular cells	High	Protein (IHC)	HPA →
Breast	Glandular cells	Medium	Protein (IHC)	HPA →
Endometrium	Cells in endometrial stroma	Medium	Protein (IHC)	HPA →
Fallopian tube	Glandular cells	Medium	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Adipose tissue	Adipocytes	Not detected	Protein (IHC)	HPA →
Adrenal gland	Glandular cells	Not detected	Protein (IHC)	HPA →
Appendix	Glandular cells	Not detected	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	Not detected	Protein (IHC)	HPA →
Bronchus	Respiratory epithelial cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced AR IHC Tips

Troubleshoot AR staining in paraffin sections by checking retrieval, compartment, cell type and scoring against documented tissue patterns.

How should I retrieve AR when nuclear staining is weak?

Use Tris-EDTA pH 9.0 heat-induced epitope retrieval at 95–98 °C for 20 min (page retrieval setting: nuclear antigen). Cool sections consistently before blocking, and compare runs using the same heating vessel and timing (standard IHC practice). Check an epididymis or seminal vesicle glandular-cell control for strong nuclear staining alongside the test section (HPA tissue IHC: High in these cells; nuclear expression in reproductive organs). If staining remains weak, compare a shorter heating time or a citrate pH 6.0 retrieval on matched sections as troubleshooting alternatives (standard IHC practice). Score nuclear signal and tissue damage together; stronger diffuse staining alone does not establish better AR detection (HPA tissue IHC: predominantly nuclear profile).

Could fixation explain weak or uneven AR staining?

AR-specific sensitivity to fixative type or fixation duration is unknown from the supplied evidence; the selected image identifies paraffin-embedded prostate carcinoma but does not state its fixative (catalog caption: M00542-1). Record fixative, time to fixation, duration and section age for each specimen before comparing staining (standard IHC practice). On matched sections, keep retrieval at Tris-EDTA pH 9.0, 95–98 °C for 20 min while checking whether weak signal follows a processing batch (page retrieval setting: nuclear antigen). Include a concurrently processed positive tissue control and inspect morphology for damaged or detached areas (standard IHC practice). Do not assign a fixation effect to AR from its phosphorylation sites or cellular distribution alone (UniProt P10275: modified residues; nucleus and cytoplasm).

What should I make of cytoplasmic AR staining in a paraffin section?

Score nuclear and cytoplasmic staining separately because AR can occupy both compartments (UniProt P10275: nucleus and cytoplasm). Ligand binding can move AR from predominantly cytoplasmic to nuclear, while RACK1 can support nuclear entry without ligand (UniProt P10275: subcellular location). In tissue IHC, nuclear expression predominates in several tissues, particularly reproductive organs (HPA tissue IHC: nuclear expression profile). Check whether nuclei in epididymis or seminal vesicle glandular cells stain on the same run before interpreting isolated cytoplasmic signal (HPA tissue IHC: High in these cells). Confirm that the chromogen lies within intact cells and compare a no-primary control when widespread cytoplasmic colour obscures compartment boundaries (standard IHC practice).

Could epitope choice explain discordant AR staining between antibodies?

Compare each antibody’s disclosed immunogen and epitope before treating discordant staining as an isoform difference (standard IHC practice). AR has 4 listed isoforms and a ligand-binding domain at residues 669–900, so epitope placement matters when assessing which forms an antibody could recognize (UniProt P10275: isoforms and NR LBD). The record also lists phosphorylation at residues including 83, 96 and 258; these annotations alone do not establish that a particular IHC epitope is masked (UniProt P10275: modified residues). Run antibodies on adjacent sections with identical retrieval and compare cellular and nuclear patterns (standard IHC practice). Report epitope coverage as unknown if the antibody documentation does not identify it (standard IHC practice).

How can I assess AR by IF alongside a cell-type marker?

For the separate IF/ICC workflow, multiplex AR with a validated marker of the expected cell type and inspect nuclear overlap with a DNA counterstain (standard IF practice; HPA tissue IHC: glandular-cell staining in epididymis and seminal vesicle). Choose spectrally separated fluorophores and place the weaker signal in a channel with low measured tissue autofluorescence (standard IF practice). Include single-stain and no-primary controls to check bleed-through and background (standard IF practice). AR has no transmembrane segment and localizes to cytoplasm and nucleus, so permeabilize cells sufficiently to access its intracellular epitope, then verify nuclear morphology is preserved (UniProt P10275: topology and subcellular location; standard IF practice). IF localization may vary by context: HPA reports supported cytosolic localization in its cell images (HPA subcellular: Cytosol).

How do I reduce diffuse brown staining without losing nuclear AR signal?

First compare the test section with a no-primary control to locate background from detection reagents or tissue components (standard chromogenic IHC practice). Apply a peroxidase block before chromogenic detection and check whether DAB deposits persist in the control (standard chromogenic IHC practice). Titrate the primary antibody and detection reagent on matched sections, keeping retrieval at Tris-EDTA pH 9.0, 95–98 °C for 20 min during that comparison (standard IHC practice; page retrieval setting: nuclear antigen). Avoid judging a darker counterstain as stronger AR; inspect crisp nuclear boundaries at the same microscope settings (HPA tissue IHC: nuclear expression profile; standard IHC practice). Retain a positive glandular-cell control so background reduction does not erase expected signal (HPA tissue IHC: High in epididymis and seminal vesicle glandular cells).

How should I quantify AR staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and region of interest before scoring, then count only intact, interpretable nuclei for the primary AR readout (standard IHC practice; HPA tissue IHC: nuclear expression profile). Report the percentage of positive nuclei and, where intensity matters, an H-score calculated from the percentages at 0, 1+, 2+ and 3+ intensity (standard IHC practice). Normalize positive counts to all eligible cells in the same compartment and region; if reporting spatial density, use positive nuclei per mm² of viable tissue (standard IHC practice). Record cytoplasmic staining separately because AR distribution can change with ligand context (UniProt P10275: subcellular location). Apply identical thresholds, counterstain and exclusion rules across sections and include a staining control on each run (standard IHC practice).

Which AR patterns support a true positive rather than staining artefact?

A convincing tissue result shows well-defined nuclear staining in the expected cells, such as epididymis or seminal vesicle glandular cells (HPA tissue IHC: High in these cells; nuclear expression profile). Treat staining restricted to cut edges, folds or necrotic areas cautiously and compare it with intact tissue away from those regions (standard IHC practice). Check a no-primary section for endogenous peroxidase or other detection background before calling diffuse brown deposits positive (standard chromogenic IHC practice). Isolated cytoplasmic staining requires context because AR can be cytoplasmic, although the supplied tissue IHC profile is mainly nuclear (UniProt P10275: subcellular location; HPA tissue IHC: nuclear expression profile). Compare cell identity and compartment with a positive control rather than interpreting colour intensity alone (standard IHC practice).

Boster reagents

Best AR / Androgen receptor IHC Antibodies

Catalog anti-AR antibodies have paraffin-section IHC images from human prostate or prostate carcinoma and an IF/ICC image from T47D cells (catalog image captions). Listed reactivity spans human, mouse and rat (catalog reactivity).

Real IHC data Immunohistochemical analysis of paraffin-embedded human prostate carcinoma, using Androgen Receptor Antibody.

Anti-Androgen Receptor AR Rabbit Monoclonal Antibody

Cat # M00542-1

View product →

Real IHC data Immunohistochemical analysis of paraffin-embedded human prostate, using Androgen Receptor Antibody.

Anti-Androgen Receptor AR Rabbit Monoclonal Antibody

Cat # M00542-2

View product →

Real IHC data Immunohistochemical analysis of paraffin-embedded human prostate carcinoma, using Androgen Receptor(AR-V7 specific) Antibody.

Anti-Androgen Receptor (AR-V7 specific) Rabbit Monoclonal Antibody

Cat # M00542-3

View product →

Real IF data

Anti-Androgen Receptor/AR Antibody ®

Cat # A00542

View product →

M00542-1 shows IHC on paraffin-embedded human prostate carcinoma, and M00542-2 shows IHC on paraffin-embedded human prostate (catalog IHC captions). M00542-3 shows IHC on paraffin-embedded human prostate carcinoma with an AR-V7-specific antibody, while A00542 shows IF/ICC in T47D cells (catalog titles and image captions).

Which to pick: For tissue IHC, choose M00542-1 for human prostate carcinoma or M00542-2 for human prostate; both are rabbit monoclonals with paraffin-section images, and their IHC starting dilutions are 1:50–1:100 and 1:50, respectively (catalog titles, IHC captions and dilutions). For AR-V7 IHC, M00542-3 has a paraffin-embedded human prostate carcinoma image and a 1:50 IHC dilution; the IHC captions do not report the fixative (catalog title, IHC caption and dilution). For IF/ICC, choose A00542, which has a T47D IF/ICC image; M00542-2 lists human, mouse and rat reactivity, although its IHC image documents human tissue only (catalog image caption, applications and reactivity).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

UniProt Consortium. UniProt entry P10275 (ANDR_HUMAN, Androgen receptor).
Human Protein Atlas. AR tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. AR subcellular location (ICC-IF): Localized to the cytosol..
Human Protein Atlas. AR antibody validation summary (3 antibodies).
Gender- and Grade-Dependent Activation of Androgen Receptor Signaling in Adult-Type Diffuse Gliomas: Epigenetic Insights from a Retrospective Cohort Study. Biomedicines 2025 — PMC12561669.
Chromosome X aneusomy and androgen receptor gene copy number aberrations in apocrine carcinoma of the breast. Virchows Archiv : an international journal of pathology 2021 — PMC8364532.
Immunohistochemical Characterization of the Androgen Receptor in Breast Cancer and Its Relationship with Breast Cancer Subtypes. Medical sciences (Basel, Switzerland) 2026 — PMC13515587.
PIK3CA mutations in androgen receptor-positive triple negative breast cancer confer sensitivity to the combination of PI3K and androgen receptor inhibitors. Breast cancer research : BCR 2014 — PMC4187324.
PubMed PMID:3216866 — UniProt-cited evidence.
PubMed PMID:3174628 — UniProt-cited evidence.
PubMed PMID:2911578 — UniProt-cited evidence.