This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
TOX / Thymocyte selection-associated high mobility group box protein TOX · IHC design guide
Design Immunohistochemistry for TOX
Plan TOX IHC-P around nuclear staining in subsets of immune cells (HPA tissue IHC). Use the catalog antibody at 2–5 μg/ml (datasheet A08441-2), with cell-type-aware controls and nuclear scoring (standard IHC practice).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
TOX 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 | Nuclear staining in subsets of immune cells (HPA tissue IHC) | |
| Staining pattern | A subset of immune cells shows nuclear staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A08441-2) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Collecting ducts and Sertoli cells also stain (HPA tissue IHC) | |
| Regulation | High in tumor-infiltrating CD8+ T cells (UniProt) | |
| Isoform / epitope | No isoforms or processing annotated; one 1–526 chain (UniProt) |
Section 1
Recommended TOX IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: A08441-2) is accompanied by three published TOX IHC protocols (PMC10518132; PMC8774984; PMC4374776).
| Sample | Paraffin-embedded human intestinal cancer tissue; fixative not specified (datasheet A08441-2) |
| Fixation | Image fixative and duration unreported (datasheet A08441-2); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A08441-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A08441-2) |
| Primary antibody | Rabbit anti-TOX, 2-5 μg/ml (datasheet A08441-2) |
| Primary incubation | Overnight at 4 °C (datasheet A08441-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A08441-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | TOX-positive staining in lymphoid tissue of appendix (HPA tissue IHC: High). HPA tissue profile: Nuclear expression mainly in subset of immune cells. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA at pH 8.0: it matches the catalog protocol (datasheet: A08441-2) and one published TOX IHC method (PMC8774984).
Section 2
What Is the Expected TOX Staining Pattern?
TOX is a nuclear protein with no transmembrane segment (UniProt O94900). In paraffin-section IHC, expect staining mainly in a subset of immune cells (HPA tissue IHC). HPA also reports high staining in kidney collecting ducts and testis Sertoli cells (HPA tissue IHC). The tissue IHC profile is rated Enhanced, with medium consistency between antibody staining and RNA expression (HPA tissue IHC).
| Distinct nuclear staining in some lymphoid cells, with adjacent cells unstained. | This fits the reported subset pattern (HPA tissue IHC) and nuclear location (UniProt O94900). Compare cells within the same section; a mixed positive and negative population can be expected. |
| Predominantly cytoplasmic or membranous staining. | This conflicts with the reported nuclear location (UniProt O94900). Treat it as a possible artefact; check morphology, background and detection controls before interpreting it as TOX. |
| Strong staining across a cell population reported as undetected. | Adipocytes and cardiomyocytes are reported as not detected (HPA tissue IHC). Check cell identity and consider cross-reactivity or endogenous detection activity (general IHC practice); these controls do not prove antibody specificity. |
| Diffuse colour over tissue and spaces between cells, without clear nuclear borders. | This is difficult to score as nuclear TOX (UniProt O94900). Check blocking, washes and the detection-only control for nonspecific colour development (general IHC practice). |
| No nuclear signal in a known-positive compartment. | Germinal center cells in tonsil and lymph node are reported High (HPA tissue IHC). Review the run controls and staining workflow (general IHC practice) before calling that compartment TOX-negative. |
💡Expected TOX appearanceCall a result positive when identifiable nuclei stain in the relevant cells, often as a subset in lymphoid tissue (UniProt O94900; HPA tissue IHC); strong diffuse cytoplasmic colour is suspect.
| Cell identity and tissue compartment | High staining is reported in tonsil and lymph-node germinal center cells, appendix lymphoid tissue, kidney collecting ducts and testis Sertoli cells (HPA tissue IHC). Score the named compartment, not the whole tissue. |
| Reported low or absent staining | Hippocampal neuronal cells are Low, while adipocytes are Not detected (HPA tissue IHC). These are cell-specific reference patterns; an unstained field elsewhere is not automatically a valid negative control. |
| Protein location and processing | TOX is nuclear, has no transmembrane segment or signal peptide, and is annotated as one 1–526 chain (UniProt O94900). An extracellular staining pattern has no support from those annotations. |
| IHC evidence and its limit | HPA rates tissue IHC Enhanced and describes medium staining–RNA consistency (HPA tissue IHC). HPA018322 is IHC Enhanced (HPA antibodies); neither rating establishes target-specific fixation sensitivity. |
| IF/ICC Q&A: where should signal appear? | Nucleoplasm and nucleoli fibrillar center are approved IF/ICC locations (HPA subcellular). Those finer locations are IF/ICC observations; assess chromogenic IHC primarily for nuclear staining (HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Tonsil germinal center cells show no signal. | This conflicts with their reported High staining (HPA tissue IHC); the cause cannot be assigned from the slide alone. | Confirm the compartment, then review retrieval, antibody dilution, detection and run controls (general IHC practice). |
| Only faint nuclear staining appears in a positive compartment. | Signal may be hard to separate from counterstain (general IHC practice); HPA levels describe observed cells, not a required intensity for every run (HPA tissue IHC). | Compare the positive compartment with adjacent cells and a matched control; review detection and counterstain (general IHC practice). |
| Colour is widespread, including areas without recognizable nuclei. | Diffuse staining does not match nuclear TOX (UniProt O94900) and may reflect background (general IHC practice). | Inspect a detection-only control; review blocking, washes and colour-development time (general IHC practice). |
| Cytoplasmic staining dominates while nuclei remain clear. | The compartment conflicts with UniProt's nuclear assignment (UniProt O94900); morphology or nonspecific binding may complicate interpretation (general IHC practice). | Verify nuclear boundaries with the counterstain and compare with a positive control before scoring (general IHC practice). |
| Adipocytes or cardiomyocytes stain strongly. | Both cell types are reported as Not detected (HPA tissue IHC); cross-reactivity or endogenous detection activity is possible (general IHC practice). | Confirm cell identity and examine the detection-only control; use an independent antibody if available (general IHC practice). |
| A lymphoid section shows scattered positive nuclei rather than uniform staining. | HPA describes nuclear expression mainly in a subset of immune cells (HPA tissue IHC); a patchy pattern alone need not indicate a failed run. | Score identifiable cells and compartments, and compare with a reported positive control such as tonsil germinal center cells (HPA tissue IHC). |
Experiment evidence
IHC & IF images showing TOX staining
A08441-2 · IHCIHC analysis of TOX using anti-TOX antibody (A08441-2). TOX was detected in a paraffin-embedded section of human intestinal cancer tissue. H
A08441-2 · IHCIHC analysis of TOX using anti-TOX antibody (A08441-2). TOX was detected in a paraffin-embedded section of mouse lymphaden tissue. Heat medi
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000198846-TOX/tissue/cerebral+cortex#imid_4807028
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000198846-TOX/tissue/colon#imid_4806954
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000198846-TOX/tissue/liver#imid_4806955
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000198846-TOX/tissue/kidney#imid_4806956
HPA · ICC-IF Rh30Human Protein Atlas immunofluorescence, Rh30, antibody HPA073241; green = TOX, blue = nucleus, red = microtubules.
HPA · ICC-IF Rh30Human Protein Atlas immunofluorescence, Rh30, antibody HPA073241; green = TOX, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for TOX IHC & IF
🧪Run tonsil first; germinal center cells should show nuclear TOX staining (HPA: High in tonsil germinal center cells; UniProt O94900: nucleus). Use adipose tissue as the negative tissue, where adipocytes are not detected for TOX (HPA: Not detected in adipocytes); cells on the tonsil slide without nuclear signal should remain at background, without assigning them an unsupported cell identity.
Positive control tissue: Appendix (Lymphoid tissue, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show TOX in Rh30, U2OS, with annotated localisation: Nucleoplasm (approved), Nucleoli fibrillar center (approved) (HPA subcellular).
Technical controls: Include no-primary (secondary-only), host- and clonality-matched rabbit IgG isotype, and TOX-knockout biological controls where available (caption: rabbit anti-TOX primary; standard IHC controls). Block endogenous peroxidase and check tonsil background before chromogenic detection (caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A08441-2 tissue-IHC caption does not state a fixative. The paraffin-section example used heat-mediated EDTA retrieval at pH 8.0, but the evidence does not establish that this retrieval condition is required (A08441-2 tissue-IHC caption). Frozen sections and IF/ICC are not shown to be easier than paraffin IHC; for tonsil, endogenous peroxidase can produce chromogenic background (HPA: High in tonsil germinal center cells; caption: HRP/DAB detection; standard IHC practice).
HPA tissue IHC evidence for TOX
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — 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 |
|---|---|---|---|---|
| Appendix | Lymphoid tissue | High | Protein (IHC) | HPA → |
| Kidney | Collecting ducts | High | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | High | Protein (IHC) | HPA → |
| Testis | Sertoli cells | High | Protein (IHC) | HPA → |
| Tonsil | Germinal center cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced TOX IHC Tips
Troubleshoot TOX staining in paraffin section IHC by checking retrieval, nuclear localisation, background, and cell specific scoring (datasheet A08441-2; UniProt O94900: nucleus).
What should I change if TOX nuclear staining is weak after retrieval?
Start with heat mediated retrieval in EDTA at pH 8.0 for paraffin TOX IHC (datasheet A08441-2). If nuclear signal is weak, compare heating exposure on adjacent sections while watching for tissue detachment or distorted morphology (standard IHC practice). Keep the catalog antibody at 2 μg/ml and its overnight 4°C incubation constant, so retrieval is the planned variable (datasheet A08441-2; standard IHC practice). Assess signal in lymph node or tonsil germinal center cells alongside a no-primary control; the expected TOX signal is nuclear (HPA: High in germinal center cells; UniProt O94900: nucleus; standard IHC practice).
Could fixation explain different TOX staining between paraffin blocks?
The selected tissue image documents a paraffin section but does not report its fixative, so TOX specific fixation sensitivity remains unknown (datasheet A08441-2). Record fixative, fixation duration, processing history, and section thickness for each specimen, then compare matched sections using EDTA pH 8.0 retrieval and 2 μg/ml primary antibody (datasheet A08441-2; standard IHC practice). If signal differs across blocks, inspect morphology and internal nuclear staining before changing retrieval or antibody concentration (standard IHC practice; HPA: nuclear expression mainly in a subset of immune cells). Establish any fixation effect with a matched fixation experiment before attributing a staining difference to TOX sensitivity (standard IHC practice).
How should I assess cytoplasmic staining with a TOX antibody?
Score TOX primarily in nuclei, because UniProt assigns it to the nucleus and HPA reports nuclear expression mainly in a subset of immune cells (UniProt O94900: nucleus; HPA: tissue profile). HPA further localises TOX to nucleoplasm and nucleoli fibrillar centers in its subcellular assessment (HPA: approved subcellular locations). If chromogenic signal appears predominantly cytoplasmic, compare the same section with a no-primary control and inspect the nuclear counterstain before calling those cells positive (standard IHC practice). TOX has no annotated transmembrane segment, so diffuse membrane staining is especially difficult to reconcile with the supplied localisation evidence (UniProt O94900: topology and subcellular location).
Can this stain distinguish a TOX isoform or a modified epitope?
The supplied UniProt record lists 0 isoforms and 0 annotated glycosylation sites or modified residues; these annotations do not establish what the catalog antibody recognises (UniProt O94900: isoforms and PTMs). Treat an apparent staining difference as an assay observation until the antibody epitope and its behaviour in processed sections are established (standard IHC practice). Compare serial sections using the documented EDTA pH 8.0 retrieval and 2 μg/ml antibody concentration before testing a retrieval change (datasheet A08441-2; standard IHC practice). If a proposed epitope specific interpretation matters, seek direct antibody specificity evidence instead of assigning the signal to an unannotated TOX variant (standard IHC practice; UniProt O94900: isoforms).
How can I follow up paraffin TOX IHC with multiplex immunofluorescence?
Use the paraffin IHC result to select regions for IF comparison, while treating IF conditions as a separate assay to optimise (datasheet A08441-2: tissue IHC; standard IF practice). Pair nuclear TOX signal with a CD8 marker when examining the reported tumor infiltrating CD8 positive T cell population, and check whether signals occupy the expected cells (UniProt O94900: protein expression in tumor infiltrating CD8 positive T cells; standard IF practice). Choose fluorophores after inspecting tissue autofluorescence, placing the weaker channel in a cleaner spectral range (standard IF practice). Optimise permeabilisation for access to TOX's nuclear epitope, since TOX has no transmembrane segment (UniProt O94900: nucleus and topology; standard IF practice).
How do I separate true TOX staining from chromogenic background?
The selected paraffin IHC example used 10% goat serum block, 2 μg/ml primary antibody overnight at 4°C, and DAB detection (datasheet A08441-2). Include a no-primary section and apply a peroxidase block when using HRP and DAB, then compare diffuse deposits with intact nuclear staining (standard IHC practice). If background persists, check washing, reagent exposure, and tissue damage before increasing antibody dilution, changing one condition at a time (standard IHC practice). A useful positive comparison is lymph node or tonsil germinal center cells; adipocytes provide a reported undetected cell population, subject to specimen quality (HPA: High in germinal center cells; HPA: adipocytes not detected; standard IHC practice).
What is a reproducible way to quantify TOX in tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region before scoring, because HPA reports nuclear TOX mainly in a subset of immune cells (HPA: tissue profile; standard IHC practice). Record the percentage of positive nuclei and, when intensity is reliable, an H-score using 0–3 intensity grades multiplied by each grade's percentage, giving a 0–300 range (standard IHC scoring practice). For spatial comparisons, report positive nuclear cell density per mm² of viable analysed tissue and specify the counted cell population (standard IHC practice). Normalise against all evaluable nuclei in that population, and apply identical staining and thresholds across sections (standard IHC practice).
When is apparent TOX positivity more likely to be an artefact?
A convincing TOX result shows nuclear signal in intact cells, consistent with UniProt localisation and HPA's tissue profile (UniProt O94900: nucleus; HPA: nuclear expression mainly in a subset of immune cells). Compare the stained cell population with an appropriate reference: HPA reports high signal in lymph node germinal center cells and kidney collecting ducts, so immune identity alone cannot define positivity (HPA: tissue staining). Be cautious with predominant membrane signal, section edge staining, and deposits over necrosis, then inspect adjacent intact tissue and a no-primary control (UniProt O94900: no transmembrane segment; standard IHC practice). Residual endogenous peroxidase can produce DAB signal, making the peroxidase blocked control useful when staining is diffuse (standard IHC practice).
Boster reagents
Best TOX / Thymocyte selection-associated high mobility group box protein TOX IHC Antibodies
A08441-2 has IHC images from paraffin-embedded human intestinal cancer and mouse lymphaden tissue (IHC image captions). No IF/ICC image is provided (IF image alts).
Real IHC data
Anti-TOX Antibody ®
Cat # A08441-2
A08441-2 is listed for IHC and has paraffin-section images from human intestinal cancer and mouse lymphaden tissue (catalog applications; IHC image captions). Its listed reactivity is human and mouse, while IF/ICC is absent from its application list (catalog reactivity; catalog applications).
Which to pick: Choose A08441-2 for paraffin-section tissue IHC; its images show human and mouse samples stained with 2 μg/ml antibody after EDTA pH 8.0 retrieval (IHC image captions). It is also the cross-species choice on the evidence provided, although its clone and the samples’ fixative are unreported (catalog reactivity; catalog clone; IHC image captions). No listed SKU is validated for IF/ICC, so an IF/ICC choice cannot be made from this payload (catalog applications; IF image alts).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O94900 (TOX_HUMAN, Thymocyte selection-associated high mobility group box protein TOX).
- Human Protein Atlas. TOX tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. TOX subcellular location (ICC-IF): Localized to the nucleoplasm and nucleoli fibrillar center..
- Human Protein Atlas. TOX antibody validation summary (2 antibodies).
- TGF-β inhibition enhances anti-tumor immunity and sensitizes PD-1 blockade therapy of bone marrow-derived myofibroblasts-induced "immunotherapy resistance" tumor model. Cancer immunology, immunotherapy : CII 2026 — PMC13388872.
- TOX Outperforms FOXP3, CD4 and GATA3 in Histopathological Diagnosis of Early Mycosis Fungoides. Turk patoloji dergisi 2023 — PMC10518132.
- Immunohistochemical Expression Patterns of CD45RO, p105/p50, JAK3, TOX, and IL-17 in Early-Stage Mycosis Fungoides. Diagnostics (Basel, Switzerland) 2022 — PMC8774984.
- TOX acts an oncological role in mycosis fungoides. PloS one 2015 — PMC4374776.
- PubMed PMID:9872452 — UniProt-cited evidence.
- PubMed PMID:12168954 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for TOX (Thymocyte selection-associated high mobility group box protein TOX). Expected localisation Nuclear staining in subsets of immune cells (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A08441-2). Validated IHC antibody: A08441-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Lymphoid tissue); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human intestinal cancer tissue; fixative not specified (datasheet A08441-2); 2. Fixation: Image fixative and duration unreported (datasheet A08441-2); 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: EDTA pH 8.0 (datasheet A08441-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A08441-2); 8. Primary antibody: Rabbit anti-TOX, 2-5 μg/ml (datasheet A08441-2); 9. Primary incubation: Overnight at 4 °C (datasheet A08441-2). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Collecting ducts and Sertoli cells also stain (HPA tissue IHC); Isoform / epitope: No isoforms or processing annotated; one 1–526 chain (UniProt); Regulation: High in tumor-infiltrating CD8+ T cells (UniProt). Evidence: UniProt O94900, HPA tissue IHC · Enhanced, HPA ICC-IF · nucleoplasm and nucleoli fibrillar center., Datasheet A08441-2, PMC10518132, PMC8774984. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/TOX/immunohistochemistry-ihc-guide.