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TNFAIP6 / Tumor necrosis factor-inducible gene 6 protein · IHC design guide
Design Immunohistochemistry for TNFAIP6
Plan TNFAIP6 IHC around extracellular deposits and medium Leydig cell staining (HPA tissue IHC). The catalog antibody has an IHC dilution range of 1:100–1:300 (datasheet A30469); interpret tissue staining cautiously because its reliability is rated uncertain (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
TNFAIP6 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 | Extracellular deposits in most tissues; uncertain (HPA tissue IHC) | |
| Staining pattern | Extracellular deposits; medium Leydig cell staining (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A30469) | |
| Positive control | Testis | |
| 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) | |
| Caveat | Secreted protein may stain away from its source cells (HPA tissue IHC) | |
| Regulation | TNF-inducible (UniProt) | |
| Isoform / epitope | No isoforms reported; mature secreted chain is aa 18–277 (UniProt) |
Section 1
Recommended TNFAIP6 IHC & IF Protocols
The catalog antibody protocol (datasheet A30469) and a published breast-tissue IHC report (PMC11673214) provide starting points for TNFAIP6 staining.
| Sample | Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A30469) |
| Fixation | Image fixative and duration unreported (datasheet A30469); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Tris-EDTA pH 9.0 (datasheet A30469); 20 min, 95–100 °C (standard) |
| 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-TNFAIP6, 1:100 - 1:300 (datasheet A30469) |
| 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 | TNFAIP6-positive staining in leydig cells of testis (HPA tissue IHC: Medium). HPA tissue profile: Extracellular deposits in most tissues and expression in kidney. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated Tris-EDTA pH 9.0 retrieval for the catalog antibody (datasheet A30469); the published report does not specify retrieval (PMC11673214).
Section 2
What Is the Expected TNFAIP6 Staining Pattern?
TNFAIP6 is a secreted protein without a transmembrane segment, so extracellular deposits are the main expected tissue pattern (UniProt P98066 topology; HPA: extracellular deposits in most tissues). HPA reports medium staining in testicular Leydig cells and expression in kidney, but its tissue IHC reliability is Uncertain; neither observation establishes a universally positive cell type (HPA: Leydig cells Medium; HPA: kidney expression; HPA: reliability Uncertain).
| Extracellular staining, with medium signal around Leydig cells in a testis section (HPA: extracellular deposits; HPA: Leydig cells Medium). | This is compatible with the reported pattern, but a deposit beside a cell does not prove that cell produced TNFAIP6: the protein is secreted (UniProt P98066: Secreted). Treat the match as provisional because HPA rates tissue IHC reliability Uncertain (HPA: reliability Uncertain). |
| Predominantly crisp nuclear staining, with little extracellular signal (UniProt P98066: Secreted; HPA: extracellular deposits). | A nuclear dominant pattern conflicts with the reported location and warrants an artefact check (UniProt P98066: Secreted; HPA: extracellular deposits). Review the no primary control and detection reagents before assigning this signal to TNFAIP6 (general IHC practice). |
| Strong cell restricted staining in a type reported as not detected, such as adipocytes (HPA: adipocytes Not detected). | Check for antibody cross reactivity or endogenous detection activity (general IHC practice). The HPA result is a reference observation, not proof of absence in every specimen, and its tissue IHC reliability is Uncertain (HPA: reliability Uncertain). |
| Broad, even color over tissue and empty slide areas, obscuring cell boundaries (general IHC practice). | Consider nonspecific background from detection or blocking conditions (general IHC practice). It should not be scored as the extracellular deposits described by HPA; compare tissue with a no primary control (HPA: extracellular deposits; general IHC practice). |
| No convincing signal in a testis section expected to show medium Leydig cell staining (HPA: Leydig cells Medium). | Review tissue preservation, retrieval, antibody conditions and detection with controls (general IHC practice). A negative result does not by itself disprove TNFAIP6 expression because the reference staining has Uncertain reliability (HPA: reliability Uncertain). |
💡Expected TNFAIP6 appearanceCall a positive result only when extracellular deposits are distinguishable from background, with at most medium Leydig cell staining as a reference; strong nuclear dominant or control matched color is suspect (HPA: extracellular deposits; HPA: Leydig cells Medium; UniProt P98066: Secreted; general IHC practice).
| Secretion and matrix association (UniProt P98066: Secreted; UniProt P98066: extracellular matrix organization). | Expect signal outside cells as well as possible cell associated staining; nearby deposits cannot identify the producing cell on their own (UniProt P98066: Secreted; HPA: extracellular deposits). Score compartment and cell association separately (general IHC practice). |
| Signal peptide and mature chain (UniProt P98066: signal 1–17; chain 18–277). | The recorded protein is processed after its signal peptide (UniProt P98066: signal 1–17; chain 18–277). Without the antibody epitope, this record cannot predict whether staining detects precursor, mature protein, or both (UniProt P98066: processing; antibody epitope not supplied). |
| Tissue reference and reliability (HPA: Leydig cells Medium; HPA: reliability Uncertain). | Use testis as a tentative reference for a detectable signal, not an absolute pass or fail control (HPA: Leydig cells Medium; HPA: reliability Uncertain). HPA notes that secreted protein location can differ from RNA location (HPA: reliability description). |
| Airway evidence differs by source (UniProt P98066: airway epithelium and submucosal gland; HPA: bronchus respiratory epithelial cells Not detected). | Report the specimen and assay context when interpreting airway staining; neither source resolves the discrepancy for a particular section (UniProt P98066: tissue specificity; HPA: bronchus Not detected). Do not treat bronchus as a guaranteed negative control (HPA: reliability Uncertain). |
| Antibody validation and retrieval (HPA: HPA050884 and CAB032719 IHC Uncertain). | Neither listed antibody has an enhanced IHC validation status in the supplied record (HPA: both IHC Uncertain). No target specific antigen retrieval or fixation effect is supplied, so optimize retrieval using controlled comparisons rather than predicting a TNFAIP6 specific response (general IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| Extracellular staining is weak or absent in the testis reference section (HPA: Leydig cells Medium; HPA: extracellular deposits). | The assay may have low sensitivity, or the reference may vary; HPA rates this tissue pattern Uncertain (general IHC practice; HPA: reliability Uncertain). | Check tissue quality, detection controls and the antibody's IHC conditions; compare retrieval conditions systematically without assuming a known TNFAIP6 fixation effect (general IHC practice; target specific fixation effect not supplied). |
| Nuclei stain more strongly than extracellular spaces (UniProt P98066: Secreted; HPA: extracellular deposits). | This compartment pattern conflicts with the secreted protein annotation and may reflect nonspecific detection (UniProt P98066: Secreted; general IHC practice). | Inspect the no primary control, blocking and counterstain; withhold a positive call unless a distinct extracellular pattern remains (general IHC practice; HPA: extracellular deposits). |
| Color covers most of the section without discrete deposits (general IHC practice; HPA: extracellular deposits). | Diffuse detection background can obscure the reported extracellular pattern (general IHC practice; HPA: extracellular deposits). | Compare the no primary control, review blocking and washing, and adjust detection conditions before scoring intensity (general IHC practice). |
| Unexpected strong staining appears in adipocytes or bronchial respiratory epithelium (HPA: both Not detected). | Cross reactivity or endogenous detection activity is possible, while sample differences remain possible because HPA reliability is Uncertain (general IHC practice; HPA: reliability Uncertain). | Check no primary and detection controls, then document compartment and specimen context; interpret airway results alongside UniProt's airway protein report (general IHC practice; UniProt P98066: airway tissue specificity). |
| A kidney section stains, but the positive cell type is unclear (HPA: kidney expression). | HPA reports kidney expression without identifying a kidney cell type in this payload, and secreted protein may appear away from its source (HPA: kidney expression; UniProt P98066: Secreted). | Record the observed compartment and distribution without assigning a kidney cell source; compare with controls and the reported extracellular profile (general IHC practice; HPA: extracellular deposits). |
| Can IF/ICC use the IHC pattern as its expected cellular location? (HPA: subcellular summary Secreted). | HPA lists no ICC/IF image cell lines or main subcellular location, so a finer cellular pattern is unverified here (HPA: ICC/IF images none; HPA: main location Not available). | Use the separate IF/ICC guide for assay design; interpret any IF signal against controls and the secreted annotation without assigning an unsupported organelle location (general IF practice; UniProt P98066: Secreted). |
Experiment evidence
IHC & IF images showing TNFAIP6 staining
A30469 · IHCImmunohistochemical analysis of paraffin-embedded human tonsil. 1, Antibody was diluted at 1:200(4° overnight). 2, Tris-EDTA,pH9.0 was used
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000123610-TNFAIP6/tissue/cerebral+cortex#imid_16469427
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000123610-TNFAIP6/tissue/colon#imid_16469369
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000123610-TNFAIP6/tissue/liver#imid_16469370
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000123610-TNFAIP6/tissue/kidney#imid_16469371
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for TNFAIP6 IHC & IF
🧪Run testis first and look for staining in Leydig cells (HPA: Medium in Leydig cells). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes); non-Leydig cells on the testis section should lack specific staining if they are to serve as internal negatives, but their TNFAIP6 status needs independent validation (HPA: only Leydig cells are listed for testis).
Positive control tissue: Testis (Leydig cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for TNFAIP6; derive a cell-line control from the positive tissue's cell type (Leydig cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary (secondary-only) control, an isotype control matched to the primary antibody’s host species, immunoglobulin class and clonality, and a TNFAIP6-knockout biological negative where available (standard IHC practice). Quench endogenous peroxidase for chromogenic detection and check the testis section for background staining; block endogenous biotin if using biotin-based detection (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected paraffin-section caption does not state its fixative (catalog antibody caption: fixative unreported). The caption uses Tris-EDTA at pH 9.0 for retrieval in tonsil, but does not establish that testis staining depends on that retrieval condition (catalog antibody caption: tonsil IHC). The supplied evidence does not establish whether frozen sections or IF are easier, and it reports no testis-specific artefact (HPA: no ICC-IF image cell lines; HPA: Medium in testis Leydig cells).
HPA tissue IHC evidence for TNFAIP6
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — No tissue staining or RNA expression data available for comparison. Secreted protein, tissue location of RNA and protein is expected to differ. 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 |
|---|---|---|---|---|
| Testis | Leydig cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced TNFAIP6 IHC Tips
Troubleshooting chromogenic TNFAIP6 IHC requires attention to retrieval, extracellular staining, and controls for a secreted protein (UniProt P98066; HPA tissue IHC).
Which retrieval conditions should I start with for TNFAIP6 paraffin sections?
Start with heat-mediated Tris-EDTA retrieval at pH 9.0 for paraffin sections (datasheet A30469). The selected tonsil image used this buffer, followed by primary antibody at 1:200 overnight at 4°C; its caption does not report a heating time (datasheet A30469). If staining is weak, compare a shorter and longer heating interval on matched sections while holding antibody dilution and development time constant (standard IHC practice). Assess whether stronger retrieval improves extracellular signal without damaging tissue morphology or increasing diffuse background, and include a section processed without primary antibody (standard IHC practice; UniProt P98066 subcellular location).
How should I troubleshoot weak staining when fixation history is uncertain?
The selected image documents paraffin-embedded tonsil but does not state its fixative, so TNFAIP6-specific fixation sensitivity is unknown (datasheet A30469). Record the actual fixative and fixation duration for each specimen, then compare matched sections with the same pH 9.0 Tris-EDTA retrieval and 1:200 primary dilution (datasheet A30469; standard IHC practice). Check tissue preservation alongside signal: disrupted edges or poorly preserved extracellular material can complicate interpretation of a secreted matrix-associated protein (UniProt P98066 function and subcellular location; standard IHC practice). If fixation histories differ, report the groups separately before attributing staining differences to biological expression (standard IHC practice).
Should TNFAIP6 staining be cellular or extracellular?
Expect extracellular staining to be plausible because TNFAIP6 is secreted and participates in hyaluronan-rich matrix organization (UniProt P98066 subcellular location and function). Its precursor has a signal peptide at residues 1–17 and no transmembrane segment, so a crisp plasma-membrane-only pattern needs additional validation (UniProt P98066 processing and topology). HPA describes extracellular deposits in most tissues, but rates its tissue IHC reliability uncertain because comparative staining and RNA evidence are unavailable (HPA tissue IHC). Map deposits against intact tissue architecture and nearby cells, and compare with a no-primary control before assigning extracellular signal to a particular producing cell (standard IHC practice; HPA tissue IHC).
What could cause an epitope-dependent TNFAIP6 staining pattern?
First confirm the catalog antibody's stated immunogen or epitope before interpreting a changed pattern; the supplied image caption does not identify that region (datasheet A30469). The record lists 0 isoforms, a Link domain at residues 36–129, and a CUB domain at 135–247 (UniProt P98066 isoforms and domains). Glycosylation is annotated at residues 118 and 258, while interactions through these domains can place TNFAIP6 in extracellular complexes (UniProt P98066 glycosylation and subunit). Compare retrieval conditions and, if available, an independently validated antibody recognizing a distinct region before calling discrepant staining an isoform-specific result (standard IHC practice; UniProt P98066 isoforms).
How can I adapt this TNFAIP6 result to multiplex immunofluorescence?
Treat IF as a separate application: the selected antibody evidence is paraffin-section chromogenic IHC, with no IF validation supplied here (datasheet A30469). For multiplexing, pair TNFAIP6 with a marker of the expected cell type, such as an epithelial marker when examining the reported airway epithelial expression, while distinguishing extracellular deposits from cell bodies (UniProt P98066 tissue specificity and subcellular location; standard IF practice). Select fluorophores in channels with low measured tissue autofluorescence and verify each channel with single-stain and no-primary controls (standard IF practice). Because TNFAIP6 is secreted and lacks a transmembrane segment, membrane-side permeabilisation is not implied for extracellular epitopes; optimise permeabilisation only if a verified epitope requires intracellular access (UniProt P98066 topology and subcellular location; standard IF practice).
How can I separate TNFAIP6 deposits from nonspecific DAB staining?
Run a no-primary section and inspect where precipitate appears relative to intact extracellular structures; TNFAIP6 can occur in extracellular deposits (standard IHC practice; HPA tissue IHC). Block endogenous peroxidase before chromogenic detection, and use matched DAB development times so differences between sections remain interpretable (standard IHC practice). If staining accumulates at tissue edges, folds, or damaged regions, compare better-preserved areas and check whether the same pattern persists in the no-primary control (standard IHC practice). Titrate around the documented 1:200 primary dilution only after confirming retrieval at pH 9.0 and checking the secondary-only background (datasheet A30469; standard IHC practice).
What should I score when TNFAIP6 appears outside cells? ⚠ ANSWER MARKED FOR VERIFICATION
Define separate cellular and extracellular regions before scoring, because TNFAIP6 is secreted and HPA describes extracellular deposits (UniProt P98066 subcellular location; HPA tissue IHC). For cellular staining, record the percentage of positive cells and an H-score based on intensity; normalise counts to the number of evaluable cells in the same annotated compartment (standard IHC practice). For extracellular deposits, measure positive area or deposit density per mm² of viable tissue, using identical thresholds and excluding folds and necrosis (standard IHC practice). Keep retrieval, antibody dilution, imaging, and DAB development consistent across samples, and report the uncertain HPA tissue IHC reliability when using its pattern as context (datasheet A30469; HPA tissue IHC; standard IHC practice).
When is a TNFAIP6-positive region convincing rather than artefactual?
A convincing result can include structured extracellular staining, consistent with TNFAIP6 secretion and its role in matrix organization (UniProt P98066 subcellular location and function). Assess cell identity cautiously: UniProt reports airway epithelial and submucosal gland protein, whereas HPA rates tissue IHC reliability uncertain and reports medium staining in testis Leydig cells (UniProt P98066 tissue specificity; HPA tissue IHC). A membrane-only signal, isolated edge staining, or staining confined to necrotic areas deserves scrutiny against tissue morphology and a no-primary section (UniProt P98066 topology; standard IHC practice). Exclude endogenous peroxidase with an appropriate block and control before assigning DAB signal to TNFAIP6, then describe observed deposits separately from inferred cellular production (standard IHC practice; UniProt P98066 subcellular location).
Boster reagents
Best TNFAIP6 / Tumor necrosis factor-inducible gene 6 protein IHC Antibodies
A30469 has real IHC data from human paraffin-embedded tonsil (IHC image caption). IF/ICC applications and human/mouse reactivity are listed, without an IF figure (catalog: A30469).
Real IHC data
Anti-TSG6 Antibody
Cat # A30469
A30469 is listed for IHC, IF and ICC, with human and mouse reactivity (catalog: A30469 applications/reactivity). Its IHC figure shows human paraffin-embedded tonsil stained at 1:200 after Tris–EDTA retrieval at pH 9.0; no IF figure is supplied (A30469 IHC image caption; catalog: A30469 IF images).
Which to pick: For tissue IHC, choose A30469 because its own figure documents staining in human paraffin-embedded tonsil; the fixative is unreported (A30469 IHC image caption). For IF/ICC, A30469 lists both applications and a 1:200–1:1000 dilution range, but provides no IF figure (catalog: A30469 applications/dilution/IF images). For work involving human and mouse samples, A30469 is listed as a rabbit polyclonal reactive with both species; its IHC figure documents human tissue only (catalog: A30469 host/clonality/reactivity; A30469 IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P98066 (TSG6_HUMAN, Tumor necrosis factor-inducible gene 6 protein).
- Human Protein Atlas. TNFAIP6 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. TNFAIP6 subcellular location (ICC-IF): Secreted.
- Human Protein Atlas. TNFAIP6 antibody validation summary (2 antibodies).
- Lung Adenocarcinoma Promotes NETosis via the NPM1-TNFAIP6-CD44-SPP1 Axis. Cancers 2026 — PMC13026014.
- Genes Associated with the Immune System Affected by Ionizing Radiation and Estrogen in an Experimental Breast Cancer Model. Biology 2024 — PMC11673214.
- TNFAIP6 Promotes Gastric Carcinoma Cell Invasion via Upregulating PTX3 and Activating the Wnt/β-Catenin Signaling Pathway. Contrast media & molecular imaging 2022 — PMC9279067.
- Molecular profiling of multiple human cancers defines an inflammatory cancer-associated molecular pattern and uncovers KPNA2 as a uniform poor prognostic cancer marker. PloS one 2013 — PMC3607594.
- PubMed PMID:1730767 — UniProt-cited evidence.
- PubMed PMID:11854277 — UniProt-cited evidence.
- PubMed PMID:15815621 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for TNFAIP6 (Tumor necrosis factor-inducible gene 6 protein). Expected localisation Extracellular deposits in most tissues; uncertain (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A30469). Validated IHC antibody: A30469 at 1:100 - 1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Leydig cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A30469); 2. Fixation: Image fixative and duration unreported (datasheet A30469); 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: Tris-EDTA pH 9.0 (datasheet A30469); 20 min, 95–100 °C (standard); 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-TNFAIP6, 1:100 - 1:300 (datasheet A30469); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Secreted protein may stain away from its source cells (HPA tissue IHC); Isoform / epitope: No isoforms reported; mature secreted chain is aa 18–277 (UniProt); Regulation: TNF-inducible (UniProt). Evidence: UniProt P98066, HPA tissue IHC · Uncertain, HPA ICC-IF · Secreted, Datasheet A30469, PMC11673214. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/TNFAIP6/immunohistochemistry-ihc-guide.