This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
TFF3 / Trefoil factor 3 · IHC design guide
Design Immunohistochemistry for TFF3
Plan chromogenic TFF3 IHC on paraffin sections using intestinal goblet cells as a positive reference (HPA tissue IHC). Assess cellular and secreted staining with appropriate controls, since TFF3 is secreted (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
TFF3 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 | Secreted/extracellular; cytoplasm also annotated (UniProt) | |
| Staining pattern | GI goblet, cervical glandular and airway epithelial cells; secreted thyroid staining (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 formalin fixation consistent across paraffin sections (standard IHC practice; not target-specific) | |
| Caveat | Secreted TFF3 may stain away from producing cells (UniProt; HPA tissue IHC) | |
| Regulation | Regulation unreported in the supplied record (UniProt) | |
| Isoform / epitope | No isoforms listed; signal peptide 1–21 cleaved, leaving chain 22–80 (UniProt) |
Section 1
Recommended TFF3 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with three published TFF3 staining protocols for paraffin sections (PMC2528153; PMC7709562; PMC6174951).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A01738); 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-TFF3, 2 μg/mL (datasheet A01738) |
| 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 | TFF3-positive staining in goblet cells of appendix (HPA tissue IHC: High). HPA tissue profile: Distinct expression in mucus secreting cells in gastrointestinal tract, cervix uteri and respiratory epithelia, with secreted positivity in thyroid gland. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval); the renal protocol uses 98 °C for 20 min (PMC6174951).
Section 2
What Is the Expected TFF3 Staining Pattern?
TFF3 is a secreted protein with no transmembrane segment; UniProt also lists cytoplasmic localization (UniProt Q07654). In paraffin-section IHC, expect staining in gastrointestinal goblet cells and other mucus-secreting epithelia, including cervical glandular and respiratory epithelial cells (HPA tissue IHC). HPA rates its tissue pattern Enhanced, while noting that secretion can complicate agreement between RNA and protein locations (HPA tissue IHC).
| Strong staining in intestinal or colonic goblet cells, with neighboring cells less prominent (HPA: High in goblet cells). | This matches the reported tissue IHC pattern. Assess staining in identified cells rather than requiring every epithelial cell to stain; HPA reports High goblet-cell staining in the appendix, colon, duodenum, rectum and small intestine (HPA tissue IHC). |
| Predominantly nuclear staining in a gastrointestinal IHC section, without the expected goblet-cell pattern (HPA tissue IHC). | Treat this as discordant tissue IHC staining and investigate an artefact or unintended detection (HPA tissue IHC; general IHC practice). Nuclear fluorescence is reported separately by HPA for ICC-IF, so nuclear location alone does not settle antibody specificity across applications (HPA subcellular ICC-IF). |
| Strong staining in an unexpected cell type while goblet cells remain unstained (HPA: High in intestinal goblet cells). | Consider antibody cross-reactivity or endogenous chromogenic detection activity; inspect the negative-reagent control and compare cell identity with the HPA tissue pattern (general IHC practice; HPA tissue IHC). |
| Diffuse color over cells and surrounding tissue, with little cell-specific contrast (general IHC practice). | Interpret cautiously: background can obscure a secreted target's distribution (UniProt Q07654 secretion; general IHC practice). Compare a negative-reagent control and check blocking, washes and detection before scoring cells (general IHC practice). |
| No signal in a section expected to contain intestinal goblet cells (HPA tissue IHC). | First verify that goblet cells are present, then check the antibody's IHC validation and the staining run (general IHC practice). HPA reports High staining in several intestinal goblet-cell populations; a blank slide alone cannot establish TFF3 absence (HPA tissue IHC). |
💡Expected TFF3 appearanceCall the IHC result positive when distinct, strong staining follows identifiable gastrointestinal goblet cells (HPA: High in goblet cells); widespread cell-independent color is a background warning, while nuclear-only tissue staining needs investigation (general IHC practice; HPA tissue IHC and subcellular ICC-IF).
| Secreted protein and topology (UniProt Q07654) | TFF3 has a signal peptide and no transmembrane segment; extracellular signal can reflect secretion, so require cell-type context when interpreting chromogenic deposits (UniProt Q07654; general IHC practice). |
| Processing and epitope information (UniProt Q07654) | The signal peptide spans residues 1–21 and the listed mature chain spans 22–80 (UniProt Q07654). Epitope location is not supplied, so these boundaries cannot predict this antibody's staining or retrieval needs. |
| Tissue and antibody evidence (HPA tissue IHC; HPA antibodies) | The tissue profile is Enhanced, and HPA lists Enhanced IHC validation for HPA035464 and CAB020681 (HPA tissue IHC; HPA antibodies). Use that support for pattern comparison, without assuming it validates a different antibody. |
| Negative comparator choice (HPA tissue IHC) | HPA reports Not detected in adipocytes of adipose tissue and in adrenal glandular cells (HPA tissue IHC). These are cell-specific comparisons, not proof that every cell in either tissue must be negative. |
| IF/ICC Q: Is nuclear fluorescence expected? (HPA subcellular ICC-IF) | HPA reports approved nucleoplasm localization, with additional nucleoli localization, in ICC-IF (HPA subcellular ICC-IF). That observation answers the IF question but does not replace the tissue IHC pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| A known-positive intestinal section is blank (HPA: High in goblet cells). | Goblet cells may be absent from the sampled area, or the IHC detection run may have failed (general IHC practice). | Confirm goblet cells on the section and review staining and detection controls (general IHC practice). HPA's tissue result alone does not specify a retrieval condition or dilution. |
| The entire section has diffuse chromogenic color (general IHC practice). | Nonspecific reagent binding or endogenous detection activity can raise background (general IHC practice). | Compare negative-reagent controls; review blocking, washes and any detection-system block before judging TFF3 distribution (general IHC practice). |
| Only nuclei stain in gastrointestinal tissue IHC (HPA tissue IHC). | The result is discordant with HPA's goblet-cell tissue pattern, although HPA separately reports nuclear ICC-IF localization (HPA tissue IHC; HPA subcellular ICC-IF). | Review cell identity, controls and antibody IHC validation; do not transfer the ICC-IF localization call directly to paraffin-section IHC (HPA antibodies; general IHC practice). |
| Unexpected cells stain more strongly than goblet cells (HPA: High in goblet cells). | Cross-reactivity or endogenous detection activity is possible; secreted TFF3 can also complicate a simple cell-of-origin reading (general IHC practice; UniProt Q07654). | Compare negative-reagent controls and the HPA cell-level tissue pattern before assigning the stain to TFF3-producing cells (general IHC practice; HPA tissue IHC). |
| Extracellular color is present without convincing goblet-cell staining (UniProt Q07654 secretion). | Secretion permits protein and source-cell locations to differ, but diffuse background can produce a similar impression (UniProt Q07654; HPA tissue IHC; general IHC practice). | Check whether deposits follow tissue structures, compare controls and seek the expected goblet-cell pattern in the same run (general IHC practice; HPA tissue IHC). |
| A proposed negative comparator contains isolated stained cells (HPA tissue IHC). | HPA's Not detected entries name specific cell populations; they do not establish a whole-section negative rule (HPA tissue IHC). | Identify the stained cell type and compare it with the exact HPA entry; avoid scoring an entire tissue from a cell-specific label (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing TFF3 staining
A01738 · IHCImmunohistochemistry of TFF3 in human small intestine tissue with TFF3 antibody at 2.5 μg/mL.
A01738 · IHCImmunohistochemistry of TFF3 in human small intestine tissue with TFF3 antibody at 2 μg/ml.
A01738 · IFImmunofluorescence of TFF3 in Human Small Intestine tissue with TFF3 antibody at 20 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000160180-TFF3/tissue/cerebral+cortex#imid_20459787
HPA · Thyroid glandHuman Protein Atlas IHC, Thyroid gland. Source: https://www.proteinatlas.org/ENSG00000160180-TFF3/tissue/thyroid+gland#imid_20459731
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000160180-TFF3/tissue/colon#imid_20463449
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000160180-TFF3/tissue/liver#imid_20463450
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA035464; green = TFF3, blue = nucleus, red = microtubules.
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA035464; green = TFF3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for TFF3 IHC & IF
🧪Run colon first: goblet cells should stain strongly (HPA: High in colon goblet cells). Use adipose tissue as a negative comparator (HPA: Not detected in adipocytes); neighboring nongoblet cells on the colon slide provide an internal negative comparison, while extracellular or luminal signal may occur because TFF3 is secreted (UniProt Q07654 subcellular location).
Positive control tissue: Appendix (Goblet cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show TFF3 in MCF-7, U2OS, with annotated localisation: Nucleoplasm (approved) (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; confirm specificity with knockout tissue or a peptide block when an appropriate reagent is available (standard IHC practice). Check endogenous peroxidase signal in the colon section when using chromogenic detection (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence, and the selected A01738 small-intestine IHC caption does not report a fixative (A01738 caption). Retrieval dependence is unreported; follow the IHC-validated antibody’s retrieval instructions and compare retrieval conditions if staining is weak (standard IHC practice). There is no supplied basis to call frozen sections or IF easier; interpret colon staining with care because secreted TFF3 can appear outside goblet cells (UniProt Q07654 subcellular location; HPA: High in colon goblet cells).
HPA tissue IHC evidence for TFF3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High consistency between antibody staining and RNA expression data. At least one protein variant secreted, tissue location of RNA and protein might differ and correlation is complex.). 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 | Goblet cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cervix | Glandular cells | High | Protein (IHC) | HPA → |
| Colon | Goblet cells | High | Protein (IHC) | HPA → |
| Duodenum | Goblet 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 → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced TFF3 IHC Tips
TFF3 IHC troubleshooting centers on retrieval, secreted staining, and cell specific interpretation (UniProt Q07654; HPA tissue IHC).
How should I retrieve TFF3 in paraffin sections with weak chromogenic staining?
Start with citrate buffer at pH 6.0 for heat induced epitope retrieval at 95–98 °C for 20 min (page retrieval setting). Let sections cool in buffer, then compare the same tissue with a no primary control so retrieval related background is visible (standard IHC practice). Use goblet cells in small intestine or colon as a positive reference for the expected staining pattern (HPA: High in small intestine and colon goblet cells). If staining remains weak, compare a second retrieval condition on adjacent sections while holding antibody concentration and detection constant; excessive heating can damage tissue morphology (standard IHC practice).
Could fixation explain weak or uneven TFF3 staining?
Target specific fixation sensitivity is unknown because the supplied TFF3 tissue caption does not state a fixative (A01738 tissue IHC caption). Record the specimen fixative and fixation duration, then compare sections processed together before changing retrieval or antibody concentration (standard IHC practice). Check whether staining differs between the tissue surface and center, and assess morphology on a counterstained section; uneven processing can complicate interpretation (standard IHC practice). The catalog image used 2.5 µg/mL antibody in human small intestine, but that caption cannot establish how TFF3 responds to any particular fixative (A01738 tissue IHC caption).
Where should convincing TFF3 staining appear in tissue?
Prioritize staining associated with intestinal goblet cells and secreted material when examining gut sections (HPA: High in small intestine and colon goblet cells; HPA: mucus secreting cell profile). TFF3 is secreted and has no transmembrane segment, so extracellular staining can accompany cellular staining without implying a membrane anchored target (UniProt Q07654 subcellular location and topology). The record also lists cytoplasmic location, making cytoplasmic signal plausible when it follows the expected cell distribution (UniProt Q07654 subcellular location; HPA tissue IHC). Treat an isolated nuclear pattern cautiously in tissue: the HPA nuclear assignment comes from subcellular ICC/IF images, whereas the tissue profile emphasizes mucus secreting cells (HPA subcellular; HPA tissue IHC).
Can processing or isoforms explain a changed TFF3 staining pattern?
The supplied record lists 0 isoforms, so an isoform switch is unsupported as an explanation for variable staining (UniProt Q07654 isoforms). TFF3 has a signal peptide at residues 1–21 and a mature chain at 22–80; check which region the antibody recognizes before interpreting absent intracellular or extracellular signal (UniProt Q07654 processing; standard IHC practice). Its P type domain spans residues 30–73, and the protein can form disulfide linked homodimers (UniProt Q07654 domain and subunit). The record lists 0 glycosylation sites, so do not assign a staining difference to a documented TFF3 glycosylation site (UniProt Q07654 glycosylation).
How can I use IF to investigate an ambiguous TFF3 IHC pattern?
On a separate IF section, multiplex TFF3 with a validated goblet cell marker to test whether gut signal follows the expected cell population (HPA: High in intestinal goblet cells; standard IF practice). Select fluorophores after inspecting an unstained section, favoring channels with low tissue autofluorescence and checking each channel separately (standard IF practice). TFF3 lacks a transmembrane segment and can be secreted or cytoplasmic; use a permeabilized condition to assess intracellular epitopes and an unpermeabilized comparison when assessing accessible extracellular signal (UniProt Q07654 topology and subcellular location; standard IF practice). Interpret nuclear IF separately from chromogenic tissue staining because HPA reports nucleoplasm and nucleoli in subcellular ICC/IF images (HPA subcellular).
How do I separate TFF3 signal from chromogenic background?
Run a no primary control through the same peroxidase detection and DAB steps to reveal detection related staining (standard chromogenic IHC practice). Block endogenous peroxidase before detection and inspect unstained and counterstained sections for pigment or deposits that could resemble chromogen (standard chromogenic IHC practice). Compare background in goblet rich mucosa with tissue regions lacking the expected TFF3 cell pattern; the reference profile places strong signal in gastrointestinal mucus secreting cells (HPA tissue IHC). If diffuse color persists, adjust blocking, washing, or antibody concentration one variable at a time while retaining the positive reference and no primary control (standard IHC practice).
How should I score TFF3 when secretion blurs cell boundaries? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scoring compartment before counting: use the percentage of positive goblet cells or an H score for cell associated staining, and measure extracellular chromogen separately (HPA: intestinal goblet cell expression; UniProt Q07654: secreted; standard IHC scoring practice). Normalize positive cell counts to all evaluable cells of the same epithelial type, or report secreted signal per tissue area rather than mixing those denominators (standard IHC scoring practice). Keep threshold, illumination, counterstain, and region selection consistent across slides (standard image analysis practice). Report both staining intensity and the number of evaluable cells so differences in goblet cell abundance are visible (standard IHC scoring practice).
Which patterns support a true TFF3 positive result?
A convincing gut result places staining in goblet cells, with possible nearby secreted signal, because those cells are strongly positive and TFF3 is secreted (HPA: High in intestinal goblet cells; UniProt Q07654 subcellular location). Question staining confined to an unexpected cell type or compartment, especially if the expected goblet cell reference is negative on the same run (HPA tissue IHC; standard IHC interpretation). Check whether apparent positivity tracks section edges or necrotic areas, which can produce misleading chromogenic patterns (standard IHC practice). Signal retained in the no primary control suggests endogenous enzyme activity or detection background rather than antibody dependent TFF3 staining (standard chromogenic IHC practice).
Boster reagents
Best TFF3 / Trefoil factor 3 IHC Antibodies
A01738 has human small-intestine IHC and IF images and a HeLa ICC image (A01738 image captions); its listed reactivity covers human, mouse and rat (catalog: A01738 reactivity).
Real IHC data
Anti-Trefoil factor 3 TFF3 Antibody
Cat # A01738
A01738 is listed for IHC-P, IF and ICC in human, mouse and rat (catalog: A01738 applications/reactivity). Its captions show human small-intestine IHC and IF and HeLa ICC (A01738 image captions).
Which to pick: For tissue IHC, choose A01738 (catalog: IHC-P application); its own human small-intestine IHC captions show 2–2.5 μg/mL, but do not report the fixative (A01738 IHC image captions). For IF/ICC, A01738 has human small-intestine IF and HeLa ICC images (A01738 IF/ICC image captions). For mouse or rat samples, A01738 lists both species as reactive (catalog: A01738 reactivity); the supplied IHC/IF tissue images are from human small intestine (A01738 image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q07654 (TFF3_HUMAN, Trefoil factor 3).
- Human Protein Atlas. TFF3 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. TFF3 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the nucleoli..
- Human Protein Atlas. TFF3 antibody validation summary (2 antibodies).
- Trefoil factor 3: a novel serum marker identified by gene expression profiling in high-grade endometrial carcinomas. British journal of cancer 2008 — PMC2528153.
- The expression of trefoil factor 3 is related to histologic subtypes and invasiveness in lung adenocarcinoma. Oncology letters 2021 — PMC7709562.
- Role of TFF3 as an adjunct in the diagnosis of Barrett's esophagus using a minimally invasive esophageal sampling device-The Cytosponge(TM). Diagnostic cytopathology 2020 — PMC7075710.
- Renal expression of trefoil factor 3 mRNA in association with tubulointerstitial fibrosis in IgA nephropathy. Nephrology (Carlton, Vic.) 2018 — PMC6174951.
- PubMed PMID:9070946 — UniProt-cited evidence.
- PubMed PMID:10950923 — UniProt-cited evidence.
- PubMed PMID:10830953 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for TFF3 (Trefoil factor 3). Expected localisation Secreted/extracellular; cytoplasm also annotated (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A01738 at 2 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Goblet cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet A01738); 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-TFF3, 2 μg/mL (datasheet A01738); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep formalin fixation consistent across paraffin sections (standard IHC practice; not target-specific); Caveat: Secreted TFF3 may stain away from producing cells (UniProt; HPA tissue IHC); Isoform / epitope: No isoforms listed; signal peptide 1–21 cleaved, leaving chain 22–80 (UniProt); Regulation: Regulation unreported in the supplied record (UniProt). Evidence: UniProt Q07654, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A01738, PMC2528153, PMC7709562. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/TFF3/immunohistochemistry-ihc-guide.