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AGR3 / Anterior gradient protein 3 · IHC design guide
Design Immunohistochemistry for AGR3
Plan AGR3 chromogenic IHC in paraffin sections using the reported glandular-cell staining pattern (HPA tissue IHC). Compare staining across cell types and titrate the catalog antibody within 1:100–1:300 (datasheet).
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 | Distinct cytoplasmic and membranous staining (HPA tissue IHC) | |
| Staining pattern | Glandular cells show distinct cytoplasmic and membranous signal (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 paraffin-section fixation consistent (standard IHC practice; not target-specific) | |
| Caveat | High lung macrophage staining may complicate scoring (HPA tissue IHC) | |
| Regulation | Rises with airway differentiation (UniProt) | |
| Isoform / epitope | No isoforms; no transmembrane split; mature chain 22–166 (UniProt) |
Section 1
Recommended AGR3 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is followed by four published AGR3 IHC protocols (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 antigen retrieval); adjust to the published method for the chosen sample.
Section 2
What Is the Expected AGR3 Staining Pattern?
AGR3 is primarily an endoplasmic reticulum protein without an annotated transmembrane segment (UniProt Q8TD06 topology and subcellular location). In paraffin sections, expect cytoplasmic and sometimes membranous staining in selected epithelial cells, especially bronchial and fallopian tube ciliated cells (HPA tissue IHC: Enhanced; High in both cell types). HPA also reports high staining in lung macrophages, so identify the stained cell type before judging a field (HPA tissue IHC: High in macrophages).
| Distinct cytoplasmic staining in bronchial or fallopian tube ciliated cell bodies, with some membranous staining. | This fits the reported tissue pattern (HPA tissue IHC: High in both ciliated cell populations; cytoplasmic and membranous profile). An endoplasmic reticulum component is plausible, although chromogenic IHC alone cannot resolve that organelle (UniProt Q8TD06 subcellular location; standard IHC practice). |
| Predominantly nuclear staining, especially where expected ciliated cells show little cytoplasmic signal. | Nuclear localisation is outside the supplied AGR3 pattern (UniProt Q8TD06: endoplasmic reticulum; HPA tissue IHC: cytoplasmic and membranous). Treat it as suspect and review controls, detection background, and tissue morphology before scoring it as AGR3 (standard IHC practice). |
| Signal appears in an unexpected cell population, while expected epithelial staining is absent or weak. | Consider cross-reactivity or endogenous detection activity, but first identify the cells: lung macrophages are reported as High by HPA (HPA tissue IHC: lung macrophages). An unexpected cell type alone does not establish antibody failure; compare a negative control and the reported tissue pattern (standard IHC practice). |
| Uniform haze covers epithelium, stroma, and other structures, obscuring cell boundaries. | This does not resemble the distinct, cell-associated pattern (HPA tissue IHC: cytoplasmic and membranous in selected cells). General IHC causes include nonspecific reagent binding or detection background; assess the negative control and background before assigning staining intensity (standard IHC practice). |
| No convincing signal in bronchial or fallopian tube ciliated cells. | These are useful known-positive populations for investigating a failed run (HPA tissue IHC: High in both). Absence in one section does not prove biological loss: confirm that the relevant cells are present, then check antibody and detection controls (standard IHC practice). |
💡Expected AGR3 appearanceCall a positive result when identifiable ciliated cell bodies show distinct, strong cytoplasmic staining, possibly with a membranous component (HPA tissue IHC: High in bronchus and fallopian tube; cytoplasmic and membranous profile); nuclear-only staining or widespread haze is suspect (UniProt Q8TD06 subcellular location; standard IHC practice).
| Cell population and tissue context | Use morphology when scoring: HPA reports High staining in bronchial and fallopian tube ciliated cells, colon goblet cells, and lung macrophages (HPA tissue IHC). UniProt reports AGR3 absent from airway mucous cells; that statement should not be extended to colon goblet cells (UniProt Q8TD06 tissue specificity; HPA tissue IHC: High in colon goblet cells). |
| Compartment and topology | AGR3 has an endoplasmic reticulum assignment and no transmembrane segment (UniProt Q8TD06). HPA's membranous IHC description should be read alongside its cytoplasmic pattern; the supplied evidence does not establish AGR3 as a plasma-membrane protein (HPA tissue IHC profile; UniProt Q8TD06 topology). |
| Antibody evidence | The listed antibody, HPA053942, has Enhanced IHC validation; the tissue profile is also labelled Enhanced for consistency with RNA expression, pending external verification (HPA antibodies; HPA tissue IHC reliability). These ratings support pattern selection but do not validate every stained cell in a new specimen (standard IHC practice). |
| Processing and epitope interpretation | UniProt annotates a signal peptide at residues 1–21 and a mature chain at 22–166 (UniProt Q8TD06 processing). Because the supplied record gives no antibody epitope, it cannot predict whether this processing affects staining; avoid attributing a weak result to peptide cleavage (UniProt Q8TD06 processing; supplied antibody record). |
| IF/ICC cross-check? | HPA places AGR3 mainly in the endoplasmic reticulum, with additional vesicle localisation, and rates HPA053942 Supported for ICC (HPA subcellular ICC-IF; HPA antibodies). That can inform a localisation cross-check, but the chromogenic tissue pattern should be judged against tissue IHC evidence (HPA tissue IHC profile). |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive ciliated cells are unstained. | The expected cells may be missing from the section, or an IHC step may have failed; HPA reports High staining in bronchial and fallopian tube ciliated cells (HPA tissue IHC; standard IHC practice). | Verify ciliated-cell morphology, then review the run's positive control, antibody application, detection reagents, and counterstain (standard IHC practice). Do not infer target-specific fixation sensitivity; none is reported in the supplied sources. |
| All compartments show brown background. | Nonspecific binding or endogenous detection activity can obscure the distinct cell-associated pattern (standard IHC practice; HPA tissue IHC profile). | Inspect a reagent negative control and review blocking, washes, detection chemistry, and antibody concentration as general IHC variables (standard IHC practice). Score only signal separable from background. |
| Nuclei dominate the signal. | The supplied localisation evidence supports endoplasmic reticulum and cytoplasmic or membranous staining, not a nuclear pattern (UniProt Q8TD06 subcellular location; HPA tissue IHC profile). | Check tissue morphology and counterstain, compare negative and known-positive controls, and repeat assessment before calling nuclei AGR3-positive (standard IHC practice). |
| A lung field stains, but airway epithelial cells look negative. | HPA reports High staining in lung macrophages, while UniProt identifies AGR3 in airway ciliated cells and not airway mucous cells (HPA tissue IHC; UniProt Q8TD06 tissue specificity). | Identify stained cells morphologically and evaluate ciliated epithelium separately from macrophages; record the cell population rather than assigning one score to the whole field (standard IHC practice). |
| Colon goblet cells stain despite an expectation that mucous cells should be negative. | The UniProt absence statement concerns airway mucous cells; HPA separately reports High staining in colon goblet cells (UniProt Q8TD06 tissue specificity; HPA tissue IHC). | Keep airway and colon interpretations separate, verify goblet-cell morphology, and compare the distribution with the HPA tissue pattern (HPA tissue IHC; standard IHC practice). |
| A presumed negative control tissue shows focal staining. | HPA's Not detected calls apply to specified cell populations, such as adipocytes in adipose tissue, rather than every cell in that section (HPA tissue IHC: adipocytes Not detected). | Identify the stained cell type and compare its signal with the specified negative population and reagent control before calling the control invalid (HPA tissue IHC; standard IHC practice). |
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: ciliated cell bodies should stain (HPA: High in bronchus ciliated cells). Use adipose tissue as a negative, where adipocytes are not detected (HPA: Not detected in adipocytes); mucous cells within the bronchus slide should remain unstained (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 an immunoglobulin control matched to the primary antibody’s host species and isotype (standard IHC practice). Use a knockout specimen or a peptide-blocked primary as a biological specificity control (A05950-1 caption: peptide-blocked breast carcinoma image), and quench endogenous peroxidase for chromogenic staining of bronchus (standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected A05950-1 paraffin-section caption leaves the fixative unreported (A05950-1 caption). Antigen retrieval dependence and frozen-section performance are unreported; optimize retrieval against bronchial ciliated-cell staining (HPA: High in bronchus ciliated cells). IF/ICC can provide a localization check because HPA reports mainly endoplasmic reticulum staining with additional vesicle staining, although tissue autofluorescence requires its own background control (HPA: subcellular localization; standard IF practice).
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, and compartment before interpreting chromogenic signal.
How should I retrieve AGR3 in paraffin sections when staining is weak?
Use citrate buffer at pH 6.0 for heat induced epitope retrieval at 95–98 °C for 20 min (page retrieval rule: cytoplasmic / membrane antigen). Keep section thickness, cooling time, and detection conditions constant while comparing retrieval runs, so a change in signal can be assigned to retrieval (standard IHC practice). If staining is weak, trial EDTA at pH 9.0 on a matched section as a fallback and check whether tissue morphology deteriorates (standard IHC practice). Include a bronchus section and assess ciliated cell bodies as a reference (HPA: High in bronchial ciliated cells).
Could fixation explain inconsistent AGR3 staining across paraffin blocks?
Target specific fixation sensitivity for AGR3 is unknown from the supplied evidence; the selected A05950-1 image shows paraffin embedded breast carcinoma but does not state its fixative (selected SKU tissue IHC caption). For newly prepared material, document fixative, tissue thickness, and time in fixative, then process comparison samples together (standard IHC practice). A 10% neutral buffered formalin workflow can serve as a general paraffin IHC starting point, but its effect on AGR3 must be established experimentally (standard IHC practice). If blocks differ, compare staining in matched epithelial areas before changing retrieval or antibody concentration (standard IHC practice).
Should AGR3 staining appear at the cell surface or around the nucleus?
Look first for cytoplasmic staining compatible with an endoplasmic reticulum distribution (UniProt Q8TD06: endoplasmic reticulum; HPA subcellular: supported endoplasmic reticulum location). A perinuclear pattern can fit that expectation, while HPA also describes cytoplasmic and membranous staining in glandular cells (HPA tissue IHC: expression profile). Membranous staining alone does not establish plasma membrane localisation, because AGR3 has no annotated transmembrane segment (UniProt Q8TD06 topology). Compare the pattern with adjacent cell types and the peptide blocked image before calling unexpected nuclear or diffuse extracellular colour AGR3 specific (selected SKU tissue IHC caption; standard IHC practice).
Could AGR3 processing or epitope placement cause a false negative?
AGR3 has a 1–21 signal peptide and a mature chain spanning residues 22–166 (UniProt Q8TD06 processing). Check the catalog antibody's disclosed immunogen, if available, against that processing boundary before interpreting a negative section (UniProt Q8TD06 processing; standard IHC practice). The supplied record annotates 0 isoforms, 0 glycosylation sites, and 0 modified residues, so these annotations offer no specific explanation for variable staining (UniProt Q8TD06 annotations). Peptide blocking in the selected breast carcinoma image supports competition with the supplied peptide, but does not by itself establish specificity in every tissue (selected SKU tissue IHC caption; standard IHC practice).
How can I check the AGR3 pattern with multiplex immunofluorescence?
Treat IF/ICC as a separate assay and pair AGR3 with a validated ciliated cell marker when examining airway epithelium (UniProt Q8TD06: expression in airway ciliated cells; standard IF practice). Choose spectrally distinct fluorophores and inspect unstained tissue in each channel before assigning signal, since tissue autofluorescence can obscure a fluorophore (standard IF practice). AGR3 is associated with the endoplasmic reticulum and has no annotated transmembrane segment, so optimise permeabilisation to expose an intracellular epitope after checking the antibody's epitope information (UniProt Q8TD06 localisation and topology; standard IF practice). Use single stain and secondary only controls to assess bleed through and background (standard IF practice).
What should I change when AGR3 chromogenic staining is widespread?
First compare a no primary control with the test section to separate detection background from primary antibody dependent signal (standard IHC practice). For a peroxidase and DAB workflow, apply a peroxidase block and inspect control tissue for residual endogenous enzyme activity (standard IHC practice). If the control is clean, titrate the catalog antibody and adjust protein blocking or wash stringency while keeping retrieval fixed (standard IHC practice). Judge the resulting pattern against the reported glandular cytoplasmic and membranous profile and against cells reported as not detected, such as adipocytes (HPA tissue IHC: expression profile; HPA: adipocytes not detected).
How should I quantify AGR3 across sections with different epithelial content? ⚠ ANSWER MARKED FOR VERIFICATION
Define the epithelial compartment and scoring threshold before reviewing outcomes, then record the % of positive cells and staining intensity on the same scale for every section (standard IHC practice). An H score can combine the proportions at each intensity as 0–300, provided all samples use the same scoring rules (standard IHC practice). Normalise to the number of evaluable epithelial cells or epithelial area rather than total section area when stromal content differs (standard IHC practice). Score relevant cell classes separately, since high staining is reported in bronchial ciliated cell bodies and colon goblet cells (HPA: High in bronchial ciliated cells and colon goblet cells).
How do I distinguish genuine AGR3 signal from staining artefact?
A credible pattern should follow the expected cell population and chiefly cytoplasmic distribution, with possible membranous staining in glandular cells (UniProt Q8TD06: airway ciliated cell expression and endoplasmic reticulum localisation; HPA tissue IHC: expression profile). Treat isolated nuclear colour, tissue edge accentuation, and necrotic deposits cautiously, and inspect a no primary control for endogenous enzyme signal (standard IHC practice). In airway tissue, compare ciliated cell bodies with mucous cells, where AGR3 was reported absent (UniProt Q8TD06: airway tissue specificity). Peptide blocking of the selected carcinoma image is supporting evidence for that image, while a second validated approach is needed to resolve discordant specimens (selected SKU tissue IHC caption; standard IHC practice).
Boster reagents
Best AGR3 / Anterior gradient protein 3 IHC Antibodies
The catalog antibody has IHC data from paraffin-embedded human breast carcinoma tissue and IF data from COS7 cells (catalog image captions); listed reactivity covers 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 image of paraffin-embedded human breast carcinoma tissue and a peptide-blocked comparator (A05950-1 IHC image caption). The same SKU lists IF/ICC applications and has an IF image of COS7 cells with a peptide-blocked comparator (A05950-1 applications; A05950-1 IF image caption).
Which to pick: Choose A05950-1 for paraffin-section IHC based on its own breast carcinoma image; the fixative is unreported (A05950-1 IHC image caption). Choose A05950-1 for IF/ICC because both applications are listed and its IF image shows COS7 cells (A05950-1 applications; A05950-1 IF image caption). For human or mouse samples, A05950-1 is the listed rabbit polyclonal option, although the supplied IHC image shows human tissue only (A05950-1 host and clonality; A05950-1 reactivity; A05950-1 IHC image 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 Distinct cytoplasmic and membranous 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 paraffin-section fixation consistent (standard IHC practice; not target-specific); Caveat: High lung macrophage staining may complicate scoring (HPA tissue IHC); Isoform / epitope: No isoforms; no transmembrane split; mature chain 22–166 (UniProt); Regulation: Rises with airway 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.