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AGR3 / Anterior gradient protein 3 · IHC design guide
Design Immunohistochemistry for AGR3
Plan AGR3 chromogenic IHC on paraffin sections using bronchial ciliated cells as a positive control (HPA tissue IHC). This guide covers the catalog antibody’s 1:100–1:300 IHC dilution range (datasheet) and cytoplasmic and membranous scoring (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
AGR3 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 and membranous tissue staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic and membranous staining in glandular cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| 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 fixation consistent across paraffin sections (standard IHC practice; not target-specific); AGR3-specific effects are unknown. | |
| Caveat | Lung macrophages may complicate epithelial scoring (HPA tissue IHC) | |
| Regulation | Increases with airway epithelial differentiation (UniProt) | |
| Isoform / epitope | No annotated isoforms; signal peptide 1–21 is removed (UniProt) |
Section 1
Recommended AGR3 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is followed by four published AGR3 IHC workflows (PMC4485854; PMC3095702; PMC6962492; PMC8232749).
| Sample | Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A05950-1) |
| Fixation | Image fixative and duration unreported (datasheet A05950-1); 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 citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane 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-AGR3, 1:100 - 1:300 (datasheet A05950-1) |
| 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 | AGR3-positive staining in enterocytes of appendix (HPA tissue IHC: High). HPA tissue profile: Distinct cytoplasmic and membranous expression in glandular cells in several tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval); use each paper’s conditions when reproducing its workflow.
Section 2
What Is the Expected AGR3 Staining Pattern?
AGR3 is mainly an endoplasmic reticulum protein, also found in cytoplasm, and has no transmembrane segment (UniProt Q8TD06). In paraffin IHC, expect cytoplasmic and membranous staining in selected epithelial cells, especially airway and fallopian tube ciliated cells (HPA tissue IHC: High; HPA profile). HPA rates the tissue pattern Enhanced for consistency with RNA data, pending external verification (HPA tissue IHC: reliability).
| Distinct staining in bronchial or fallopian tube ciliated cell bodies. | This matches high staining in those cells (HPA tissue IHC: High). A cytoplasmic signal fits the endoplasmic reticulum location (UniProt Q8TD06; HPA subcellular). Assess cell identity and compartment together; color alone does not establish specificity. |
| Cytoplasmic and apparent membrane staining in glandular epithelium. | This is within the reported tissue IHC profile (HPA tissue IHC: cytoplasmic and membranous). AGR3 has no annotated transmembrane segment (UniProt Q8TD06 topology), so apparent membrane staining should be interpreted as an observed IHC pattern, not proof that AGR3 spans the membrane. |
| Predominantly nuclear staining with little epithelial cytoplasmic signal. | A nuclear dominant pattern does not match the reported endoplasmic reticulum location or tissue IHC profile (UniProt Q8TD06; HPA subcellular; HPA tissue IHC). Treat it as suspect and check the detection and counterstain controls before assigning it to AGR3. |
| Staining appears in an unexpected cell population. | Check the exact tissue and cell type against HPA before calling it cross-reactivity: lung macrophages and colon goblet cells are reported High (HPA tissue IHC), while UniProt reports airway ciliated cells and no detection in mucous cells (UniProt Q8TD06). Persistent unsupported staining warrants specificity controls. |
| No signal in bronchial or fallopian tube ciliated cells. | Those cell bodies are reported High (HPA tissue IHC), so absence in an intact known-positive section raises a technical or specimen question. Confirm that the expected cells are present, then review staining-run controls; a negative result alone cannot identify the failed step. |
💡Expected AGR3 appearanceCall a section positive when selected epithelial cells show distinct cytoplasmic, sometimes apparent membranous, staining, with strong ciliated cell-body signal in bronchus or fallopian tube (HPA tissue IHC: profile; High); isolated nuclear staining is inconsistent with the reported endoplasmic reticulum location (UniProt Q8TD06; HPA subcellular).
| Cell type and tissue | Expression is uneven: bronchial and fallopian tube ciliated cells, appendix and duodenal enterocytes, and cervical glandular cells are High, whereas salivary gland glandular cells and efferent duct ciliated cells are Low (HPA tissue IHC). Specify the cell population when scoring. |
| Compartment and topology | Endoplasmic reticulum localization supports cytoplasmic staining (UniProt Q8TD06; HPA subcellular). HPA also describes a membranous tissue IHC pattern (HPA tissue IHC); with no transmembrane segment annotated, that pattern alone cannot establish surface localization (UniProt Q8TD06 topology). |
| Cell-type disagreement across sources | Colon goblet cells are High in HPA tissue IHC, while UniProt reports no detection in airway mucous cells (HPA tissue IHC; UniProt Q8TD06). These observations concern different tissues; record tissue and cell identity instead of extending either statement to all mucous cells. |
| Antibody and evidence scope | HPA053942 has Enhanced IHC validation, and the HPA tissue profile is Enhanced for staining and RNA consistency, pending external verification (HPA antibodies; HPA tissue IHC). These ratings support pattern interpretation but do not validate every new specimen or staining run. |
| Protein processing | UniProt annotates a signal peptide at residues 1–21 and a mature chain at 22–166, with no annotated glycosylation sites or isoforms (UniProt Q8TD06). The payload does not identify the antibody epitope, so processing cannot be used here to predict retrieval needs or staining strength. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive ciliated epithelium is blank. | The target cells may be absent from the section, or the staining run may have failed; HPA reports High signal in bronchial and fallopian tube ciliated cell bodies (HPA tissue IHC). | Verify the cells on the counterstained section and inspect the run's positive control; then review the routine IHC sequence, including retrieval, primary-antibody application and chromogenic detection (general IHC practice). |
| Most structures show diffuse color. | Background can obscure the selective epithelial pattern reported for AGR3 (HPA tissue IHC: profile); the appearance alone cannot distinguish nonspecific binding from detection background. | Compare with an appropriate no-primary control, then review blocking, washes and chromogen development for the run (general IHC practice). Judge AGR3 only after the expected cell populations remain discernible. |
| Signal is predominantly nuclear. | Nuclear dominance conflicts with the reported endoplasmic reticulum localization and cytoplasmic tissue pattern (UniProt Q8TD06; HPA subcellular; HPA tissue IHC). | Check nuclear counterstain and detection controls, then reassess whether cytoplasmic signal is present in HPA High ciliated cells (HPA tissue IHC; general IHC practice). Do not score nuclear color alone as AGR3. |
| Unexpected cells stain strongly. | The result may reflect a documented tissue-specific cell pattern or nonspecific signal: lung macrophages and colon goblet cells are High in HPA tissue IHC, despite UniProt's airway ciliated-cell emphasis (HPA tissue IHC; UniProt Q8TD06). | Identify the stained tissue and cells first. If the population is unsupported, compare no-primary and known-positive controls and review antibody specificity before interpreting the signal (general IHC practice). |
| Only faint signal appears in a proposed positive control. | The chosen cell type may have a low reported level: salivary gland glandular cells, efferent duct ciliated cells and placental trophoblastic cells are Low (HPA tissue IHC). | Use a section containing an HPA High cell population to assess the run, and compare like cell types when scoring specimens (HPA tissue IHC; general IHC practice). |
| Can an IF/ICC image settle an ambiguous IHC result? | HPA ICC-IF supports mainly endoplasmic reticulum localization with additional vesicles, while its tissue IHC profile describes cytoplasmic and membranous staining (HPA subcellular; HPA tissue IHC). The assays show different aspects of localization. | Use ICC-IF as compartment context only; resolve the paraffin-section result with its cell pattern and IHC controls (HPA subcellular; HPA tissue IHC; general IHC practice). IF/ICC procedures belong in the separate guide. |
Experiment evidence
IHC & IF images showing AGR3 staining
A05950-1 · IHCImmunohistochemistry analysis of paraffin-embedded human breast carcinoma tissue, using AGR3 Antibody. The picture on the right is blocked w
A05950-1 · IFImmunofluorescence analysis of COS7 cells, using AGR3 Antibody. The picture on the right is blocked with the synthesized peptide.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000173467-AGR3/tissue/cerebral+cortex#imid_14994536
HPA · DuodenumHuman Protein Atlas IHC, Duodenum. Source: https://www.proteinatlas.org/ENSG00000173467-AGR3/tissue/duodenum#imid_14994469
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000173467-AGR3/tissue/colon#imid_14994478
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000173467-AGR3/tissue/liver#imid_14994471
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA053942; green = AGR3, blue = nucleus, red = microtubules.
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA053942; green = AGR3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for AGR3 IHC & IF
🧪Run bronchus first and expect staining in ciliated cell bodies (HPA: High in bronchus ciliated cells). Run adipose tissue as a negative and expect adipocytes to be unstained (HPA: Not detected in adipocytes); mucous cells on the bronchus slide provide an internal negative comparison (UniProt Q8TD06: not detected in airway mucous cells).
Positive control tissue: Appendix (Enterocytes, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show AGR3 in A-549, RPTEC/TERT1, U2OS, with annotated localisation: Endoplasmic reticulum (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a control IgG matched to the primary antibody’s host species and clonality (standard IHC practice). Use knockout material or a peptide-block control as a biological negative (selected-SKU tissue-IHC caption: staining blocked with synthesized peptide), and check endogenous peroxidase signal on the bronchus slide during chromogenic detection (standard IHC practice).
⚠️Feasibility: Paraffin-section IHC is documented for the selected catalog antibody, but its tissue-IHC caption does not report a fixative (selected-SKU tissue-IHC caption). A target-specific fixation window, fixation effect, and antigen-retrieval dependency are unreported in the supplied evidence; the caption does not establish fixation (selected-SKU tissue-IHC caption). The evidence does not establish whether frozen sections or IF are easier; if using IF on bronchus, assess tissue autofluorescence with an unstained section (standard IF practice). The selected A05950-1 tissue-IHC caption documents paraffin sections, but does not specify the fixative (selected-SKU IHC image A05950-1).
HPA tissue IHC evidence for AGR3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High consistency between antibody staining and RNA expression data. Pending external verification.). 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 | Enterocytes | High | Protein (IHC) | HPA → |
| Bronchus | Ciliated cells (cell body) | High | Protein (IHC) | HPA → |
| Cervix | Glandular cells | High | Protein (IHC) | HPA → |
| Colon | Goblet cells | High | Protein (IHC) | HPA → |
| Duodenum | Enterocytes | 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 AGR3 IHC Tips
Troubleshoot AGR3 staining in paraffin sections by checking retrieval, cell type, compartment, controls, and scoring before interpreting signal.
What retrieval should I start with for AGR3 in paraffin sections?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval rule: cytoplasmic / membrane antigen). Allow sections to cool in buffer, then compare signal in a positive control with background in a negative control under the same detection conditions (standard IHC practice). If staining remains weak, test a second retrieval condition on serial sections while holding antibody concentration and development time constant (standard IHC practice). AGR3 is mainly associated with the endoplasmic reticulum, so assess intracellular staining in the expected cells rather than judging retrieval by surface staining alone (UniProt Q8TD06; HPA subcellular).
Could fixation explain weak or uneven AGR3 staining?
AGR3-specific sensitivity to fixation is unknown from the supplied evidence; the selected A05950-1 image describes paraffin-embedded breast carcinoma but does not state the fixative (A05950-1 tissue-IHC caption). Record the fixative and processing history for each specimen before comparing staining intensity across cases (standard IHC practice). If fixation varies, stain matched control sections in the same run and inspect whether weak areas track tissue edges or processing differences (standard IHC practice). Optimise retrieval on representative sections before changing antibody concentration, and report any processing differences alongside the AGR3 score (standard IHC practice).
Where should convincing AGR3 signal appear in tissue?
Look for predominantly cytoplasmic staining, with an endoplasmic reticulum pattern where cellular detail permits (UniProt Q8TD06 subcellular; HPA subcellular). HPA also describes cytoplasmic and membranous staining in glandular cells and an additional vesicular location (HPA tissue IHC; HPA subcellular). AGR3 has no annotated transmembrane segment, so a membrane-like outline alone does not establish plasma-membrane localisation (UniProt Q8TD06 topology). Compare the same cell population at high magnification with its neighbouring cells and counterstain; diffuse extracellular colour or staining confined to tissue edges warrants checking the chromogenic workflow (standard IHC practice).
How should the AGR3 epitope affect antibody selection and controls?
Check the antibody's stated immunogen or mapped epitope before interpreting discordant staining, because that information is not supplied for the selected A05950-1 image (A05950-1 tissue-IHC caption). AGR3 has a signal peptide at residues 1–21 and a mature chain at 22–166; cleavage can matter if an antibody recognises the signal peptide (UniProt Q8TD06 processing). The supplied record annotates 0 isoforms and 0 glycosylation sites, so neither provides an evidenced explanation for staining differences here (UniProt Q8TD06). The selected image includes peptide blocking; use that observation alongside tissue and reagent controls, rather than treating blocking alone as proof of cellular specificity (A05950-1 tissue-IHC caption; standard IHC practice).
How can IF help investigate an ambiguous chromogenic AGR3 pattern?
Use IF as a follow-up localisation check while keeping the tissue IHC result tied to its own controls (standard IHC / IF practice). Multiplex AGR3 with a ciliated-cell marker in airway sections, where UniProt reports expression in ciliated epithelium; assess each marker in separate channels before interpreting overlap (UniProt Q8TD06 tissue specificity; standard IF practice). Choose a fluorophore channel after measuring tissue autofluorescence with an unstained section, and include single-stain controls for spectral bleed-through (standard IF practice). If the antibody epitope is intracellular, use controlled permeabilisation to reach the endoplasmic reticulum; the supplied caption does not identify the A05950-1 epitope or establish an IF protocol (HPA subcellular; A05950-1 tissue-IHC caption).
What should I check when AGR3 IHC has diffuse brown background?
Run a no-primary control and inspect whether colour follows tissue, section edges, or the entire slide before changing AGR3 antibody concentration (standard IHC practice). For peroxidase-based detection, verify the peroxide block and shorten DAB development if the control also turns brown; these are general chromogenic workflow checks (standard IHC practice). Reassess blocking and washes, then titrate the antibody using identical retrieval and detection conditions on serial sections (standard IHC practice). Because HPA reports high staining in lung macrophages as well as bronchial ciliated-cell bodies, identify the stained cell type before calling lung signal nonspecific (HPA tissue IHC).
How should I score AGR3 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored compartment and cell population before reviewing outcomes; AGR3 can show cytoplasmic and membranous patterns in glandular cells (HPA tissue IHC). For tumour epithelium, record the percentage of positive cells and staining intensity, then calculate an H-score on a 0–300 scale using the same scoring rules across sections (standard IHC practice). If the question concerns sparse positive cells, report positive-cell density per mm² of viable, evaluable tissue instead (standard IHC practice). Normalise comparisons to the relevant cell population or viable tissue area, and document excluded necrotic and damaged regions (standard IHC practice).
How do I distinguish genuine AGR3 staining from artefact?
A plausible positive result follows the expected cell type and intracellular pattern: AGR3 is reported in airway ciliated cells and mainly localises to the endoplasmic reticulum (UniProt Q8TD06 tissue specificity; HPA subcellular). Treat isolated nuclear staining or a purely extracellular deposit cautiously, because neither matches the supplied localisation evidence (UniProt Q8TD06 subcellular; HPA subcellular). Check edge-concentrated colour, necrotic areas, and no-primary controls for section artefact or endogenous enzyme activity before assigning a positive score (standard IHC practice). Interpret the A05950-1 peptide-blocked breast carcinoma image as supporting evidence for that image, while comparing your own positive and negative controls under matched conditions (A05950-1 tissue-IHC caption; standard IHC practice).
Boster reagents
Best AGR3 / Anterior gradient protein 3 IHC Antibodies
A05950-1 has IHC data in paraffin-embedded human breast carcinoma tissue and IF data in COS7 cells (catalog image captions); listed reactivity is Human and Mouse (catalog: reactivity).
Real IHC data
Anti-Anterior gradient protein 3 AGR3 Antibody
Cat # A05950-1
A05950-1 will render with an IHC figure from paraffin-embedded human breast carcinoma tissue (A05950-1 IHC caption). It is listed for IHC, IF, ICC and ELISA, with Human and Mouse reactivity; its IF image shows COS7 cells (A05950-1 catalog).
Which to pick: Choose A05950-1 for tissue IHC: its own image shows paraffin-embedded human breast carcinoma tissue, and its IHC dilution is 1:100–1:300 (A05950-1 IHC caption; datasheet: IHC dilution). The fixative is unreported (A05950-1 IHC caption). The same rabbit polyclonal antibody is listed for IF/ICC at 1:200–1:1000 and for Human and Mouse reactivity, although its pictured IF example is COS7 cells (A05950-1 catalog and IF caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8TD06 (AGR3_HUMAN, Anterior gradient protein 3).
- Human Protein Atlas. AGR3 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. AGR3 subcellular location (ICC-IF): Mainly localized to the endoplasmic reticulum. In addition localized to vesicles..
- Human Protein Atlas. AGR3 antibody validation summary (1 antibodies).
- Anterior gradient protein 3 is associated with less aggressive tumors and better outcome of breast cancer patients. OncoTargets and therapy 2015 — PMC4485854.
- The anterior gradient homolog 3 (AGR3) gene is associated with differentiation and survival in ovarian cancer. The American journal of surgical pathology 2011 — PMC3095702.
- Anterior Gradient 3 Promotes Breast Cancer Development and Chemotherapy Response. Cancer research and treatment 2020 — PMC6962492.
- AGR3 Regulates Airway Epithelial Junctions in Patients with Frequent Exacerbations of COPD. Frontiers in pharmacology 2021 — PMC8232749.
- PubMed PMID:12477722 — UniProt-cited evidence.
- PubMed PMID:12975309 — UniProt-cited evidence.
- PubMed PMID:12690205 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for AGR3 (Anterior gradient protein 3). Expected localisation Cytoplasmic and membranous tissue staining (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A05950-1 at 1:100 - 1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Enterocytes); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A05950-1); 2. Fixation: Image fixative and duration unreported (datasheet A05950-1); 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 citrate buffer, pH 6.0 , 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-AGR3, 1:100 - 1:300 (datasheet A05950-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific); AGR3-specific effects are unknown.; Caveat: Lung macrophages may complicate epithelial scoring (HPA tissue IHC); Isoform / epitope: No annotated isoforms; signal peptide 1–21 is removed (UniProt); Regulation: Increases with airway epithelial differentiation (UniProt). Evidence: UniProt Q8TD06, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the endoplasmic reticulum. In addition localized…, Datasheet A05950-1, PMC4485854, PMC3095702. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/AGR3/immunohistochemistry-ihc-guide.