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AGR2 / Anterior gradient protein 2 homolog · IHC design guide
Design Immunohistochemistry for AGR2
Plan chromogenic AGR2 IHC in paraffin sections around the cytoplasmic glandular pattern (HPA tissue IHC). Use bronchial respiratory epithelium as a positive reference and esophageal squamous epithelium as a negative reference (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
AGR2 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 | Cytoplasmic staining in glandular tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in glandular and urothelial epithelia (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A02922-2) | |
| Positive control | Appendix+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 paraffin-section fixation consistent across samples. (standard IHC practice; not target-specific) | |
| Caveat | Secreted AGR2 may extend beyond producing cells (UniProt) | |
| Regulation | Tissue-dependent abundance (UniProt) | |
| Isoform / epitope | No isoforms annotated; mature aa 21–175; no TM segment (UniProt) |
Section 1
Recommended AGR2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by four published AGR2 IHC protocols for head and neck, breast, and canine mammary tissues (PMC4496185; PMC2361240; PMC8460385; PMC8532596).
| Sample | Paraffin-embedded human colorectal adenocarcinoma tissue; fixative not specified (datasheet A02922-2) |
| Fixation | Image fixative and duration unreported (datasheet A02922-2); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A02922-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A02922-2) |
| Primary antibody | Rabbit anti-AGR2, 2-5μg/ml (datasheet A02922-2) |
| Primary incubation | Overnight at 4 °C (datasheet A02922-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A02922-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | AGR2-positive staining in endocrine cells of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in glandular tissues, including glands in GI, female and male genitalia, and urothelial epithelia. No signal in the no-primary control. |
💡Decision noteFor the catalog antibody, start with heat-mediated EDTA at pH 8.0 (datasheet A02922-2). Citrate at pH 6.0 is documented for other antibodies (PMC4496185; PMC8460385).
Section 2
What Is the Expected AGR2 Staining Pattern?
AGR2 staining should be predominantly cytoplasmic in glandular epithelia, with strong examples in cervical and endometrial glandular cells and bronchial respiratory epithelium (HPA: tissue IHC, Enhanced reliability). An endoplasmic reticulum pattern is biologically consistent with its ER and secreted localization; AGR2 has no transmembrane segment (UniProt O95994: localization and topology). HPA reports medium consistency between antibody staining and RNA expression (HPA: reliability description).
| Strong cytoplasmic staining in cervical or endometrial glandular cells, or bronchial respiratory epithelium (HPA: High). | This matches the cell types and intensity reported by HPA. Compare positive cells with adjacent structures on the same section before scoring; a tissue-wide score can conceal the cell-specific pattern (HPA: tissue IHC; general IHC practice). |
| Predominantly nuclear or sharply membrane-restricted staining, with little cytoplasmic signal. | This conflicts with the reported cytoplasmic tissue pattern and supported ER localization (HPA: tissue IHC and subcellular IF). Treat it as an unexpected result requiring controls, rather than assigning AGR2 positivity from that compartment alone (general IHC practice). |
| Strong signal in adipocytes or esophageal squamous epithelial cells (HPA: Not detected). | Those cell types are reported as unstained by HPA. Assess cross-reactivity or endogenous detection activity, especially if the expected glandular or respiratory epithelial pattern is absent; confirm with detection controls (HPA: tissue IHC; general IHC practice). |
| Uniform chromogen over many cell types, extracellular areas, or the whole section. | A broad haze does not reproduce HPA's cell-selective cytoplasmic pattern (HPA: tissue IHC). Excess detection background or nonspecific binding is plausible; judge localization against a negative detection control and tissue morphology (general IHC practice). |
| No signal in a well-preserved cervical glandular or bronchial epithelial region (HPA: High). | An expected positive cell population has failed to stain. Check whether the cells are present, then assess antibody application, detection and counterstain against a known-positive section processed in the same run (HPA: tissue IHC; general IHC practice). |
💡Expected AGR2 appearanceCall AGR2 positive when the expected epithelial cells show clear cytoplasmic staining, potentially strong in HPA High populations; isolated nuclear, membrane-only or indiscriminate tissue-wide signal needs verification (HPA: tissue IHC and subcellular IF; general IHC practice).
| Cell and tissue context | HPA reports High staining in cervical, endometrial and fallopian tube glandular cells, bronchial respiratory epithelial cells, and endocrine cells in appendix, colon and duodenum; it reports Low staining in kidney distal tubules and breast glandular cells (HPA: tissue IHC). Choose and score controls by cell type. |
| Localization and protein topology | AGR2 is secreted and ER-localized, with a signal peptide at residues 1–20 and no transmembrane segment (UniProt O95994: localization, processing and topology). Cytoplasmic staining is consistent with the tissue data; extracellular signal alone should not define a positive cell (HPA: tissue IHC; general IHC practice). |
| Evidence strength | HPA labels the tissue profile Enhanced, while describing only medium consistency between staining and RNA data. Its listed antibody, HPA007912, has Enhanced IHC validation, meaning reproduction by independent antibodies or orthogonal data (HPA: reliability and antibody validation). Interpret an unusual pattern with appropriate controls. |
| IF/ICC Q&A: where should AGR2 appear? | An ER-associated signal is the supported intracellular expectation (HPA: subcellular ICC-IF). HPA lists A-549, RT-4 and U2OS among cell lines with ICC-IF images and marks HPA007912 ICC Approved (HPA: subcellular data and antibody validation). This does not establish an IF staining protocol for this IHC guide. |
| Situation | Likely cause | Next action |
|---|---|---|
| Positive control is blank. | The expected cell population may be absent, or an IHC workflow step may have failed (HPA: High cell types; general IHC practice). | Confirm the relevant cells on the section; check primary-antibody application and detection reagents, then compare with a known-positive section from the same run (general IHC practice). |
| Signal appears mainly in nuclei or along cell borders. | That distribution is discordant with cytoplasmic tissue staining and supported ER localization (HPA: tissue IHC and subcellular IF). | Inspect morphology and detection controls; repeat with the IHC-validated antibody and evaluate whether cytoplasmic staining appears in an expected positive cell population (HPA: antibody validation; general IHC practice). |
| Adipocytes or esophageal squamous cells stain strongly. | HPA reports AGR2 as Not detected in those cells; cross-reactivity or endogenous detection activity is possible (HPA: tissue IHC; general IHC practice). | Compare a negative detection control and an expected positive cell population in the same run before accepting the signal as specific (general IHC practice). |
| Weak staining appears in kidney distal tubules or breast glands. | Low staining is reported in these cell types, so weak signal alone does not indicate a failed run (HPA: tissue IHC). | Score the defined cells against a High positive control; use the same scoring criteria across sections and avoid treating weak staining as a strong positive (HPA: tissue IHC; general IHC practice). |
| Brown haze obscures epithelial boundaries. | Background from binding or detection may hide the reported cell-selective cytoplasmic pattern (HPA: tissue IHC; general IHC practice). | Review negative detection controls and optimize routine blocking, washing or detection conditions for the assay; reassess identifiable cell boundaries afterward (general IHC practice). |
| A strong positive tissue gives patchy or unexpectedly weak staining. | Cell composition may vary across the section; HPA's High designation applies to specified cell types, and its staining-to-RNA consistency is medium (HPA: tissue IHC and reliability). | Check that the expected cells are present and intact, then compare another section or a known-positive control processed in the same run before revising the interpretation (general IHC practice). |
Experiment evidence
IHC & IF images showing AGR2 staining
A02922-2 · IHCIHC analysis of AGR2 using anti-AGR2 antibody (A02922-2). AGR2 was detected in a paraffin-embedded section of human colorectal adenocarcinom
A02922-2 · IHCIHC analysis of AGR2 using anti-AGR2 antibody (A02922-2). AGR2 was detected in a paraffin-embedded section of human non-small cell lung canc
M02922 · IHCImmunohistochemical analysis of paraffin-embedded human uterus, using AGR2 Antibody.
M02922 · IFImmunofluorescent analysis using the Antibody at 1:50 dilution.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000106541-AGR2/tissue/cerebral+cortex#imid_2718612
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000106541-AGR2/tissue/colon#imid_2718554
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000106541-AGR2/tissue/liver#imid_2718555
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000106541-AGR2/tissue/kidney#imid_2718556
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA007912; green = AGR2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA007912; green = AGR2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for AGR2 IHC & IF
🧪Run bronchus first: respiratory epithelial cells should stain strongly (HPA: High in bronchus respiratory epithelial cells); run adipose tissue as the negative tissue, where adipocytes are not detected (HPA: Not detected in adipocytes). On the bronchus slide, use adjacent nonepithelial cells as internal background comparators: they should lack the respiratory epithelial staining pattern, although their AGR2-negative status is not established by the supplied HPA rows (HPA: bronchus respiratory epithelial cells High).
Positive control tissue: Appendix (Endocrine cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show AGR2 in A-549, RT-4, U2OS, with annotated localisation: Endoplasmic reticulum (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and an isotype-matched rabbit IgG control alongside an AGR2 knockout specimen or, if the cognate peptide is available, a peptide-blocked primary control (selected-SKU caption: rabbit primary antibody). Quench endogenous peroxidase in bronchus sections before HRP/DAB detection (selected-SKU caption: HRP/DAB detection).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU paraffin-section caption does not state a fixative (selected-SKU caption: fixative not stated). Heat retrieval in EDTA at pH 8.0 was used, but retrieval dependence and any advantage of frozen sections or IF/ICC are unreported; HPA ICC-IF images support ER localization as an IF reference (selected-SKU caption: EDTA retrieval; HPA subcellular: ER supported). In bronchus, distinguish epithelial staining from luminal material when scoring because AGR2 can be secreted (UniProt O95994: Secreted; HPA: High in bronchus respiratory epithelial cells).
HPA tissue IHC evidence for AGR2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium 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 |
|---|---|---|---|---|
| Appendix | Endocrine cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cervix | Glandular cells | High | Protein (IHC) | HPA → |
| Colon | Endocrine cells | High | Protein (IHC) | HPA → |
| Duodenum | Endocrine cells | High | 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 → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced AGR2 IHC Tips
Troubleshoot AGR2 staining in paraffin sections by checking retrieval, controls, cellular distribution, and scoring before comparing specimens.
What retrieval should I use if AGR2 staining is weak in paraffin sections?
Start with heat-mediated antigen retrieval in EDTA buffer at pH 8.0 (datasheet A02922-2). The selected paraffin-section example used this retrieval before overnight incubation with 2 μg/ml primary antibody at 4°C; its fixation method was unreported (datasheet A02922-2). If staining remains weak, vary heating and cooling within your established tissue protocol while keeping a known positive section in each run (standard IHC practice). Compare signal in glandular or respiratory epithelial cells with background in adjacent tissue, and change one retrieval variable at a time so the source of improvement remains clear (HPA tissue IHC; standard IHC practice).
How can I troubleshoot variable AGR2 staining across paraffin blocks?
The selected AGR2 paraffin-section caption does not report a fixative, so target-specific fixation sensitivity is unknown (datasheet A02922-2). Record the fixative, fixation duration, tissue thickness, processing history, and section age for each block before attributing a difference to AGR2 abundance (standard IHC practice). Run sections from the compared blocks together with the same EDTA pH 8.0 retrieval and antibody incubation to reduce run-to-run variation (datasheet A02922-2; standard IHC practice). If one block stays weak, examine its morphology and a known positive control, then report the block-level limitation rather than assigning a target-specific fixation effect (standard IHC practice).
Which staining compartment should count as AGR2 positive in IHC?
Score convincing cellular cytoplasmic staining, especially in glandular tissue, against the section’s morphology (HPA tissue IHC). AGR2 is annotated in the endoplasmic reticulum and as secreted, with no transmembrane segment; a crisp plasma-membrane-only pattern therefore needs independent confirmation (UniProt O95994 localisation and topology). Cytoplasmic chromogenic signal cannot resolve endoplasmic reticulum from nearby cytoplasm at routine light-microscope resolution, so describe the observed compartment conservatively (standard IHC practice). Check whether stained cells fit the expected tissue pattern, such as bronchial respiratory epithelial cells or cervical glandular cells, and compare them with surrounding cells on the same slide (HPA tissue IHC; standard IHC practice).
Could AGR2 processing or an isoform explain discordant staining?
The supplied record lists 0 isoforms, a signal peptide at residues 1–20, and a mature chain at residues 21–175 (UniProt O95994 processing and isoforms). It also lists 0 glycosylation sites and no transmembrane segment; those annotations alone do not establish which epitope this antibody recognizes (UniProt O95994 glycosylation and topology). Check the antibody’s documented immunogen or epitope before proposing loss of a signal-peptide epitope, and keep retrieval and dilution constant during that comparison (standard IHC practice). If the epitope is undocumented, treat altered staining after processing as an open technical question and compare independent controls rather than assigning it to a specific AGR2 form (standard IHC practice).
How should I investigate AGR2 by multiplex IF alongside tissue IHC?
For a separate IF experiment, pair AGR2 with a marker that identifies the expected cell population, such as a validated glandular or respiratory epithelial marker appropriate to the section (HPA tissue IHC; standard IF practice). Choose fluorophores after inspecting unstained tissue autofluorescence, and place the weaker signal in a channel with lower background (standard IF practice). Because AGR2 has supported endoplasmic-reticulum localisation and no transmembrane segment, intracellular-epitope staining generally requires permeabilisation after fixation; confirm the antibody epitope before setting detergent conditions (HPA subcellular; UniProt O95994 topology; standard IF practice). Include single-colour and secondary-only controls to distinguish channel bleed-through, autofluorescence, and nonspecific signal from cellular colocalisation (standard IF practice).
How do I reduce diffuse brown background without losing AGR2 signal?
Start by separating tissue pigment and precipitated chromogen from cellular staining using an unstained section and a primary-antibody omission control (standard IHC practice). For HRP–DAB detection, include an endogenous peroxidase block and prepare the chromogen according to the detection system’s instructions; these are general IHC steps, not AGR2-specific findings (standard IHC practice). The selected AGR2 example used 10% goat serum blocking and 2 μg/ml primary antibody overnight at 4°C (datasheet A02922-2). If background persists, titrate primary antibody and detection exposure while retaining the same positive control, and judge improvement by cellular signal relative to nearby negative areas (standard IHC practice).
What is a defensible way to score AGR2 IHC across samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the eligible cell population before scoring, because HPA reports cytoplasmic expression in glandular tissues and different levels among cell types (HPA tissue IHC). For each region, record the percentage of eligible cells at intensity 0–3 and calculate an H-score as the sum of percentage times intensity, with a possible range of 0–300 (standard IHC practice). Normalise positive-cell counts or stained area to eligible cells or viable tissue area, rather than total section area when tissue composition varies (standard IHC practice). Apply one threshold and imaging setup across batches, and report background controls and excluded necrotic or damaged regions alongside the scores (standard IHC practice).
When should an apparent AGR2-positive area be treated as artefact?
Give greatest weight to reproducible cellular cytoplasmic signal in the expected epithelial or glandular population, interpreted with tissue morphology (HPA tissue IHC; standard IHC practice). Question isolated nuclear or sharply membrane-only staining because supported endoplasmic-reticulum localisation and the absence of a transmembrane segment do not predict those patterns as the sole signal (HPA subcellular; UniProt O95994 topology). Compare section edges, folds, and necrotic areas with intact interior tissue, since these regions can produce misleading chromogenic intensity (standard IHC practice). A primary-antibody omission control helps identify endogenous enzyme or detection-system colour; interpret weak results cautiously because HPA rates tissue IHC reliability as Enhanced with medium RNA–staining consistency (standard IHC practice; HPA tissue IHC).
Boster reagents
Best AGR2 / Anterior gradient protein 2 homolog IHC Antibodies
Both anti-AGR2 antibodies have human paraffin-section IHC images (catalog captions). M02922 also has an IF image (catalog); A02922-2 lists human, mouse, and rat reactivity (catalog).
Real IHC data
Anti-Anterior Gradient 2/AGR2 Antibody ®
Cat # A02922-2
Real IHC data
Anti-AGR2/Ag 2 Rabbit Monoclonal Antibody
Cat # M02922
A02922-2 shows IHC staining in paraffin sections of human colorectal adenocarcinoma, non-small cell lung cancer, and bladder urothelial carcinoma with squamous differentiation (A02922-2 IHC captions). M02922 shows IHC staining in paraffin-embedded human uterus and has an IF image with no sample identified (M02922 IHC/IF captions).
Which to pick: For tissue IHC, A02922-2 provides documented retrieval with EDTA at pH 8.0 in the illustrated human tumor sections; M02922 is a human-reactive monoclonal option illustrated in paraffin-embedded uterus (A02922-2 IHC captions; M02922 catalog and IHC caption). For IF/ICC, M02922 has an IF image at 1:50, while A02922-2 lists IF/ICC at 5 μg/ml without an IF image (M02922 IF caption; A02922-2 catalog). For mouse or rat samples, A02922-2 lists reactivity, though its IHC images show human samples only; the fixative is unreported in both antibodies’ IHC captions (A02922-2 catalog and IHC captions; M02922 IHC caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O95994 (AGR2_HUMAN, Anterior gradient protein 2 homolog).
- Human Protein Atlas. AGR2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. AGR2 subcellular location (ICC-IF): Localized to the endoplasmic reticulum..
- Human Protein Atlas. AGR2 antibody validation summary (1 antibodies).
- Anterior gradient protein 2 expression in high grade head and neck squamous cell carcinoma correlated with cancer stem cell and epithelial mesenchymal transition. Oncotarget 2015 — PMC4496185.
- Significance of the metastasis-inducing protein AGR2 for outcome in hormonally treated breast cancer patients. British journal of cancer 2006 — PMC2361240.
- The Utility of SOX2 and AGR2 Biomarkers as Early Predictors of Tamoxifen Resistance in ER-Positive Breast Cancer Patients. International journal of surgical oncology 2021 — PMC8460385.
- Serum Level of Tumor-Overexpressed AGR2 Is Significantly Associated with Unfavorable Prognosis of Canine Malignant Mammary Tumors. Animals : an open access journal from MDPI 2021 — PMC8532596.
- PubMed PMID:9790916 — UniProt-cited evidence.
- PubMed PMID:12975309 — UniProt-cited evidence.
- PubMed PMID:12853948 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for AGR2 (Anterior gradient protein 2 homolog). Expected localisation Cytoplasmic staining in glandular tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A02922-2). Validated IHC antibody: A02922-2 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Endocrine cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colorectal adenocarcinoma tissue; fixative not specified (datasheet A02922-2); 2. Fixation: Image fixative and duration unreported (datasheet A02922-2); 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 retrieval: EDTA pH 8.0 (datasheet A02922-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A02922-2); 8. Primary antibody: Rabbit anti-AGR2, 2-5μg/ml (datasheet A02922-2); 9. Primary incubation: Overnight at 4 °C (datasheet A02922-2). Watch for: Fixation: Keep paraffin-section fixation consistent across samples. (standard IHC practice; not target-specific); Caveat: Secreted AGR2 may extend beyond producing cells (UniProt); Isoform / epitope: No isoforms annotated; mature aa 21–175 ; no TM segment (UniProt); Regulation: Tissue-dependent abundance (UniProt). Evidence: UniProt O95994, HPA tissue IHC · Enhanced, HPA ICC-IF · endoplasmic reticulum., Datasheet A02922-2, PMC4496185, PMC2361240. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/AGR2/immunohistochemistry-ihc-guide.