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TRAF6 / TNF receptor-associated factor 6 · IHC design guide
Design Immunohistochemistry for TRAF6
Plan chromogenic TRAF6 IHC in paraffin sections around the cytoplasmic staining observed in most tissues (HPA tissue IHC). This guide covers fixation, staining controls and interpretation of tissue variation (standard IHC practice).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
TRAF6 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic staining in most tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in most tissues, including glandular cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M00185-1) | |
| Caveat | Lung macrophages show low staining (HPA tissue IHC) | |
| Regulation | Expression regulation unreported (UniProt) | |
| Isoform / epitope | No alternate isoforms listed; 1–522 chain (UniProt) |
Section 1
Recommended TRAF6 IHC & IF Protocols
The catalog antibody protocol is followed by published TRAF6 IHC methods for glioblastoma, rat intestine, and liver tissues (PMC9685220; PMC4005622; PMC5041287).
| Sample | Paraffin-embedded Rat ovary tissue; fixative not specified (datasheet M00185-1) |
| Fixation | Image fixative and duration unreported (datasheet M00185-1); 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 Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear 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 monoclonal (clone BOF-20) anti-TRAF6, 1:50-1:200 (datasheet M00185-1) |
| 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 | TRAF6-positive staining in glandular cells of adrenal gland (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with pH 9 HIER at 95–98 °C for 20 min (page retrieval); the glioblastoma protocol uses a closely matching condition (PMC9685220).
Section 2
What Is the Expected TRAF6 Staining Pattern?
TRAF6 is chiefly cytoplasmic in tissue IHC, with medium staining reported in several glandular, hematopoietic, neuronal and endothelial cell populations (HPA tissue IHC: Approved; medium RNA–staining consistency). Cytoplasmic staining is consistent with a protein without a transmembrane segment (UniProt Q9Y4K3: topology). Nuclear localisation is also possible in specific lymphocyte contexts (UniProt Q9Y4K3: subcellular location).
| Cytoplasmic staining in the expected cells, with tissue architecture and nuclei still clear. | This matches the broad tissue pattern (HPA tissue IHC: cytoplasmic expression in most tissues). Medium staining is documented in bone marrow hematopoietic cells, cerebral cortex neuronal cells and colon endothelial cells (HPA tissue IHC). Judge the named cell population, since a tissue-wide positive or negative label would hide that distinction (general IHC interpretation). |
| Staining is confined to cell borders or extracellular spaces, while cytoplasm is clear. | An exclusively membranous or extracellular pattern conflicts with the reported predominant cytoplasmic IHC pattern (HPA tissue IHC) and lack of a transmembrane segment (UniProt Q9Y4K3: topology). Investigate nonspecific staining or detection artefact (general IHC practice). Do not automatically reject nuclear staining: UniProt reports TRAF6 in nuclei of some lymphocytes (UniProt Q9Y4K3: subcellular location). |
| An unexpected cell population stains more strongly than the documented positive population. | Check cell identity and the detection controls before assigning TRAF6 expression (general IHC interpretation). Cross-reactivity or endogenous detection activity can produce misleading chromogenic signal (general IHC practice). The HPA observations identify particular stained cells; they do not establish that every other cell type must be negative (HPA tissue IHC: observed cell patterns). |
| Colour spreads across the section, obscuring cell boundaries and compartments. | Diffuse background prevents a reliable localisation call (general IHC interpretation). In chromogenic IHC, incomplete blocking, concentrated primary antibody, inadequate washing or endogenous enzyme activity can contribute (general IHC practice). Compare a control processed without primary antibody before interpreting weak cytoplasmic colour (general IHC practice). |
| No signal appears in a documented positive cell population. | A technical failure is possible, particularly if the expected cells are present but unstained (general IHC practice). Recheck the staining run against a documented example: medium staining is reported in bone marrow hematopoietic cells and breast glandular cells (HPA tissue IHC). HPA calls its tissue IHC reliability Approved with medium RNA–staining consistency, so one negative section does not settle biological absence (HPA tissue IHC). |
💡Expected TRAF6 appearanceA persuasive positive is cell-associated, predominantly cytoplasmic chromogen in a documented population, such as medium-stained bone marrow hematopoietic cells (HPA tissue IHC); diffuse, extracellular or exclusively border staining warrants a false-positive check (general IHC interpretation; UniProt Q9Y4K3: topology).
| Tissue and cell population | HPA reports low tissue specificity and cytoplasmic expression in most tissues, but its examples name particular positive cells (HPA tissue IHC). Select and score the cell population, rather than treating every cell in a positive tissue as equivalent (general IHC interpretation). |
| Strength of the reference pattern | The supplied positive examples are Medium, while lung macrophages, bronchus respiratory epithelial cells and several other populations are Low (HPA tissue IHC). Weak staining in a Low population needs a readable control; do not use it alone to set a strong-positive threshold (general IHC interpretation). |
| Compartment and topology | UniProt lists cytoplasm, cell cortex, nucleus and lipid droplet; HPA tissue IHC describes mainly cytoplasmic staining (UniProt Q9Y4K3: subcellular location; HPA tissue IHC). No transmembrane segment or signal peptide is reported (UniProt Q9Y4K3: topology and processing). These records do not define an IHC epitope or a required antigen-retrieval condition. |
| Antibody evidence | Three listed antibodies have Approved IHC status: HPA019805, HPA020599 and CAB004605 (HPA antibodies: IHC). Approved status and medium RNA–staining consistency support using the observed pattern as a reference, while leaving an individual specimen open to control-based interpretation (HPA tissue IHC: reliability; general IHC interpretation). |
| IF/ICC Q: Should a mitochondrial or nucleolar image determine the IHC call? | A: No. HPA ICC-IF reports mainly mitochondrial and additional nucleolar localisation, both marked uncertain (HPA subcellular ICC-IF). Tissue IHC instead reports cytoplasmic expression in most tissues (HPA tissue IHC). Assess the paraffin-section chromogenic result against the tissue IHC pattern; the IF/ICC guide covers that application separately. |
| Situation | Likely cause | Next action |
|---|---|---|
| No cytoplasmic signal in a documented medium-positive population. | The staining run, antibody conditions or tissue handling may have failed (general IHC practice); HPA medium staining is an observed reference, not a guarantee for every section (HPA tissue IHC). | Confirm the named cells are present; review run controls, primary-antibody dilution, antigen-retrieval settings and detection reagents using the chosen antibody's IHC instructions (general IHC practice). |
| Only weak colour is seen in lung macrophages. | Low staining is reported for this population (HPA tissue IHC: lung macrophages, Low); weak colour alone is hard to separate from background (general IHC interpretation). | Compare it with a documented medium-positive cell population and the run's background controls before scoring it positive (HPA tissue IHC; general IHC practice). |
| The whole section is brown, including spaces between cells. | Diffuse chromogen can reflect nonspecific binding, excess reagent or endogenous detection activity (general IHC practice). It does not match cell-associated cytoplasmic expression (HPA tissue IHC). | Inspect the no-primary control, blocking and washes; reassess antibody concentration and detection conditions according to the chosen IHC workflow (general IHC practice). |
| Signal appears only at cell borders or outside cells. | That distribution conflicts with the predominant cytoplasmic tissue pattern (HPA tissue IHC) and the absence of a transmembrane segment (UniProt Q9Y4K3: topology). | Check section edges and control slides for nonspecific chromogen; score TRAF6 only where cell-associated staining and cell identity can be assessed (general IHC interpretation). |
| Unexpected nuclei stain in a lymphocyte-rich area. | Nuclear TRAF6 can occur in resting and activated T- and B-lymphocytes (UniProt Q9Y4K3: subcellular location); nuclear staining alone therefore cannot establish artefact. | Verify the stained cell population and compare cytoplasmic staining and controls on the same run before accepting or rejecting the nuclear result (general IHC interpretation). |
| A sample disagrees with the HPA tissue pattern. | The HPA tissue profile is Approved but has medium consistency between antibody staining and RNA expression (HPA tissue IHC: reliability). A single discordant specimen has several possible biological or technical explanations (general IHC interpretation). | Recheck cell identity, run controls and compartment; if the discrepancy persists, compare staining with another IHC Approved antibody from the supplied HPA list (HPA antibodies: IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing TRAF6 staining
M00185-1 · IHCImmunohistochemical analysis of paraffin-embedded Rat ovary, using the Antibody at 1:50 dilution.
M00185-1 · IHCImmunohistochemical analysis of paraffin-embedded Rat stomach, using the Antibody at 1:50 dilution.
M00185 · IHCImmunohistochemical analysis of paraffin-embedded human lung carcinoma, using TRAF6 Antibody.
A00185-1 · IFImmunocytochemistry of TRAF6 in K562 cells with TRAF6 antibody at 0.5 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000175104-TRAF6/tissue/cerebral+cortex#imid_1573628
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000175104-TRAF6/tissue/colon#imid_1573554
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000175104-TRAF6/tissue/liver#imid_1573563
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000175104-TRAF6/tissue/kidney#imid_1573556
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA019805; green = TRAF6, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA019805; green = TRAF6, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for TRAF6 IHC & IF
🧪Run breast first: its glandular cells should show medium staining (HPA: breast glandular cells, Medium). HPA detects TRAF6 in all 45 scored tissues, so there is no validated negative tissue; use no-primary and isotype controls, and treat unstained cells within the breast section as a background reference rather than confirmed TRAF6-negative cells (HPA: no negative rows).
Positive control tissue: Adrenal gland (Glandular cells, HPA Medium)
Negative control tissue: None in HPA: TRAF6 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show TRAF6 in A-431, U-251MG, U2OS, with annotated localisation: Mitochondria (uncertain) (HPA subcellular).
Technical controls: Run a no-primary, secondary-only slide and an isotype control matched to the primary antibody’s host species, clonality, and concentration; use TRAF6 knockout material as a biological specificity control where available (standard IHC practice). For chromogenic detection in breast tissue, block endogenous peroxidase and, if using biotin-based detection, assess endogenous biotin background (standard IHC practice).
⚠️Feasibility: A TRAF6-specific fixation window and the effect of antigen retrieval are unreported in the supplied evidence; optimize retrieval for the chosen antibody (supplied target/application evidence; standard IHC practice). The selected M00185-1 caption shows paraffin-section rat ovary staining at 1:50, but its fixative is unreported (selected tissue-IHC caption). The supplied evidence does not establish whether frozen sections or IF are easier; breast background from endogenous peroxidase or biotin should be checked with the detection controls (standard IHC practice).
HPA tissue IHC evidence for TRAF6
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: TRAF6 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced TRAF6 IHC Tips
TRAF6 staining is predominantly cytoplasmic across tissues, with context dependent nuclear localisation reported (HPA tissue IHC; UniProt Q9Y4K3 localisation).
How should I optimise retrieval when TRAF6 staining is weak in paraffin sections?
Start with Tris-EDTA pH 9.0 heat induced epitope retrieval at 95–98 °C for 20 min (page retrieval rule). After cooling, compare a positive control section with a no primary control while keeping antibody dilution and chromogen development constant (standard IHC practice). If staining remains weak, test a shorter or longer retrieval time on adjacent sections and check whether tissue morphology or background deteriorates (standard IHC practice). Expect predominantly cytoplasmic staining across tissues; nuclear staining can occur in specified lymphocyte contexts, so do not use nuclear intensity alone to select retrieval conditions (HPA tissue IHC; UniProt Q9Y4K3 localisation).
How can I assess whether fixation is masking TRAF6 in my sections?
Target specific fixation sensitivity is unknown here, and the rat ovary caption reports paraffin embedding without stating a fixative (selected IHC caption, M00185-1). Record the fixative and fixation duration for each specimen, then compare sections processed under documented conditions using the same retrieval and staining run (standard IHC practice). Include a known staining control and a no primary control so weak signal can be distinguished from failed detection or diffuse background (standard IHC practice). If fixation histories differ, interpret intensity comparisons cautiously and assess preservation of cellular detail before assigning a biological difference (standard IHC practice).
Which TRAF6 staining compartments should I expect in tissue IHC?
Use cytoplasmic staining as the main tissue benchmark because the tissue survey reports cytoplasmic expression in most tissues (HPA tissue IHC). Nuclear signal may be plausible in lymphocytes, since TRAF6 has been reported in nuclei of resting and activated T and B lymphocytes and in punctate nuclear complexes (UniProt Q9Y4K3 localisation). The cell imaging survey instead lists uncertain mitochondrial and nucleolar localisation, so treat matching puncta as a hypothesis for follow up, not proof of organelle identity in tissue (HPA subcellular). Compare adjacent cell populations, morphology and control sections before interpreting an unusual compartment as TRAF6 (standard IHC practice).
How do I investigate unexpected TRAF6 staining when the antibody epitope is unclear?
The supplied record lists one 522 amino acid TRAF6 chain and 0 isoforms, so an isoform explanation is unsupported by this evidence (UniProt Q9Y4K3 processing and isoforms). TRAF6 has no transmembrane segment and contains a MATH domain at residues 350–499, but the antibody epitope is not provided (UniProt Q9Y4K3 topology and domains; supplied antibody evidence). Check the antibody documentation for its immunogen region before attributing staining differences to epitope accessibility (standard IHC practice). If the pattern persists, compare independently validated epitopes or an orthogonal expression measure, with cell identity and compartment recorded for each section (standard IHC practice).
How should I adapt TRAF6 localisation checks to multiplex IF?
For the separate IF workflow, pair TRAF6 with CD45 when examining hematopoietic cells, a population showing medium tissue staining in bone marrow (HPA tissue IHC; standard IF practice). Choose a fluorophore channel with low tissue autofluorescence and inspect unstained and single stain controls before assigning faint puncta to TRAF6 (standard IF practice). TRAF6 has no transmembrane segment and is reported in cytoplasm and nucleus, so use controlled permeabilisation to expose intracellular epitopes while preserving cell boundaries (UniProt Q9Y4K3 topology and localisation; standard IF practice). Interpret mitochondrial or nucleolar overlap cautiously because those cell imaging assignments are uncertain (HPA subcellular).
What should I check when chromogenic TRAF6 staining looks diffuse?
Run a no primary control and inspect tissue edges, damaged areas and pigment before assigning diffuse colour to TRAF6 (standard IHC practice). For a peroxidase based chromogenic workflow, verify endogenous peroxidase blocking, appropriate protein blocking, washes and matched chromogen development across sections (standard IHC practice). Titrate the primary antibody on adjacent sections; the selected paraffin embedded rat ovary image used 1:50, which documents that image rather than a universal working dilution (selected IHC caption, M00185-1). Compare the resulting cellular pattern with predominantly cytoplasmic tissue staining and retain morphology as a check on interpretation (HPA tissue IHC; standard IHC practice).
How should I score TRAF6 IHC across regions with different cell mixtures? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, then record the percentage of positive cells and an intensity weighted H-score within that population (standard IHC practice). Report cytoplasmic and nuclear scores separately, because cytoplasmic expression predominates in tissue while nuclear localisation has been reported in particular lymphocyte contexts (HPA tissue IHC; UniProt Q9Y4K3 localisation). For spatial counts, report positive cells per mm² of viable analysed tissue and normalise comparisons to the relevant cell population rather than total mixed cells (standard IHC practice). Keep retrieval, detection and threshold settings consistent, and exclude folds, necrosis and section edges from the measured area (standard IHC practice).
How can I distinguish genuine TRAF6 signal from staining artefacts?
Give greatest weight to cellular cytoplasmic staining that follows intact morphology, since that is the predominant tissue pattern reported for TRAF6 (HPA tissue IHC; standard IHC practice). Evaluate nuclear staining in its cell context: lymphocyte nuclear localisation is reported, whereas an isolated nuclear edge pattern needs technical review (UniProt Q9Y4K3 localisation; standard IHC practice). Check suspicious signal against no primary controls, especially at section edges, necrotic areas and sites of possible endogenous peroxidase activity (standard IHC practice). The tissue survey has approved reliability with medium agreement between staining and RNA, so use cell level controls or orthogonal evidence before making a strong biological claim from intensity alone (HPA tissue IHC; standard IHC practice).
Boster reagents
Best TRAF6 / TNF receptor-associated factor 6 IHC Antibodies
Anti-TRAF6 antibodies have IHC images from paraffin-embedded rat and human tissues and ICC/IF images from K562 cells (catalog image captions).
Real IHC data
Anti-TRAF6 Rabbit Monoclonal Antibody
Cat # M00185-1
Real IHC data
Anti-TRAF6 Rabbit Monoclonal Antibody
Cat # M00185
Real IF data
Anti-TRAF6 Antibody
Cat # A00185-1
M00185-1 has IHC images from paraffin-embedded rat ovary, rat stomach, human prostate cancer and human astrocytoma; M00185 has an IHC image from paraffin-embedded human lung carcinoma (catalog image captions). A00185-1 has ICC and IF images from K562 cells (catalog image captions).
Which to pick: For tissue IHC, choose rabbit monoclonal M00185-1 for its 1:50–1:200 IHC range and pictured rat paraffin sections, or rabbit monoclonal M00185 for its pictured human paraffin section and 1:50 IHC dilution (catalog host, dilution and IHC image captions). For IF/ICC, choose M00185 or A00185-1; both list those applications, and A00185-1 has pictured K562 cells (catalog applications and image captions). For cross-species work, all three list Human, Mouse and Rat reactivity; the M00185-1 and M00185 IHC captions identify paraffin-embedded samples but do not report a fixative (catalog reactivity and IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9Y4K3 (TRAF6_HUMAN, TNF receptor-associated factor 6).
- Human Protein Atlas. TRAF6 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. TRAF6 subcellular location (ICC-IF): Mainly localized to the mitochondria. In addition localized to the nucleoli..
- Human Protein Atlas. TRAF6 antibody validation summary (3 antibodies).
- Comparing tumor microRNA profiles of patients with long‑ and short‑term‑surviving glioblastoma. Molecular medicine reports 2023 — PMC9685220.
- TRAF6 promotes chemoresistance to paclitaxel of triple negative breast cancer via regulating PKM2-mediated glycolysis. Cancer medicine 2023 — PMC10587986.
- Glutamine attenuates the inhibitory effect of methotrexate on TLR signaling during intestinal chemotherapy-induced mucositis in a rat. Nutrition & metabolism 2014 — PMC4005622.
- Relationship between TRAF6 and deterioration of HCC: an immunohistochemical and in vitro study. Cancer cell international 2016 — PMC5041287.
- PubMed PMID:8837778 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:16554811 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for TRAF6 (TNF receptor-associated factor 6). Expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: M00185-1 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded Rat ovary tissue; fixative not specified (datasheet M00185-1); 2. Fixation: Image fixative and duration unreported (datasheet M00185-1); 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 Tris-EDTA buffer, pH 9.0 , 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 monoclonal (clone BOF-20) anti-TRAF6, 1:50-1:200 (datasheet M00185-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M00185-1); Caveat: Lung macrophages show low staining (HPA tissue IHC); Isoform / epitope: No alternate isoforms listed; 1–522 chain (UniProt); Regulation: Expression regulation unreported (UniProt). Evidence: UniProt Q9Y4K3, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the mitochondria. In addition localized to the…, Datasheet M00185-1, PMC9685220, PMC4005622. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/TRAF6/immunohistochemistry-ihc-guide.