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TBX21 / T-box transcription factor TBX21 · IHC design guide
Design Immunohistochemistry for TBX21
Plan TBX21 chromogenic IHC in paraffin sections around nuclear staining in a subset of immune cells (HPA tissue IHC). Use spleen as a positive tissue reference (HPA tissue IHC) and score nuclear staining within the relevant cell population.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
TBX21 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 a subset of immune cells (HPA tissue IHC) | |
| Staining pattern | Selective staining of immune-cell nuclei (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Bone marrow+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
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 M00404) | |
| Caveat | Kidney proximal tubules also show medium staining (HPA tissue IHC) | |
| Regulation | T-cell-specific expression (UniProt) | |
| Isoform / epitope | No annotated isoforms; one 1–535 chain (UniProt) |
Section 1
Recommended TBX21 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is supplemented by one published TBX21 IHC workflow (PMC10948251).
| Sample | Paraffin-embedded human spleen tissue; fixative not specified (datasheet M00404) |
| Fixation | Image fixative and duration unreported (datasheet M00404); 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 AFHO-20) anti-TBX21, 1:50-1:200 (datasheet M00404) |
| 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 | TBX21-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: Medium). HPA tissue profile: Selective nuclear expression in a subset of immune cells. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 retrieval at 95–98 °C for 20 min (page antigen-retrieval rule for nuclear TBX21; UniProt: nucleus).
Section 2
What Is the Expected TBX21 Staining Pattern?
TBX21 is nuclear and lacks a transmembrane segment (UniProt Q9UL17 topology). Expect selective nuclear staining in a subset of immune cells (HPA tissue IHC: Enhanced reliability), consistent with T-cell specificity (UniProt Q9UL17). HPA also reports staining in kidney proximal tubules (HPA tissue IHC).
| Nuclear stain in lymph-node non-germinal center cells (HPA tissue IHC). | Expected selective pattern; reported level is medium (HPA tissue IHC). |
| Predominantly cytoplasmic stain. | Suspect nonspecific signal; TBX21 is nuclear (UniProt Q9UL17; HPA tissue IHC). |
| Adipocytes or bronchial respiratory epithelium stain. | Suspect cross-reactivity or endogenous activity; both are not detected (HPA tissue IHC). |
| Diffuse stain across the section. | Suspect background rather than selective nuclear staining (HPA tissue IHC). |
| No stain in lymph-node non-germinal center cells. | Check assay performance; medium staining is reported there (HPA tissue IHC). |
💡Expected TBX21 appearanceMedium nuclear stain in lymph-node non-germinal center cells; cytoplasmic stain is suspect (HPA tissue IHC; UniProt Q9UL17).
| Cell distribution (HPA tissue IHC) | Immune-cell staining is selective; kidney proximal tubules also show medium stain (HPA tissue IHC). |
| Antibody validation (HPA antibodies) | CAB009524 and CAB030043 are IHC Enhanced (HPA antibodies). |
| Processing (UniProt Q9UL17) | One chain spans residues 1–535; no signal peptide or propeptide (UniProt Q9UL17). |
| Situation | Likely cause | Next action |
|---|---|---|
| No nuclear signal in lymph node. | Expected cells show medium stain (HPA tissue IHC). | Check a positive control, retrieval, primary incubation and detection (standard IHC practice). |
| Strong cytoplasmic stain. | Expected location is nuclear (UniProt Q9UL17; HPA tissue IHC). | Check the counterstain and a primary-omission control (standard IHC practice). |
| Diffuse background. | Blocking or detection conditions may contribute (standard IHC practice). | Optimize blocking and primary concentration (standard IHC practice). |
| Adipocytes or bronchial epithelium stain. | These cells are not detected by HPA tissue IHC; suspect nonspecific or endogenous signal. | Use primary-omission controls; check endogenous peroxidase (standard IHC practice). |
| Kidney proximal tubules stain. | HPA reports medium staining there (HPA tissue IHC). | Score this compartment against the HPA tissue IHC pattern. |
| What IF/ICC pattern is established? | No ICC-IF images or main location are available (HPA subcellular). | Use the separate IF/ICC guide; do not infer an IF pattern from IHC. |
Experiment evidence
IHC & IF images showing TBX21 staining
M00404 · IHCImmunohistochemical analysis of paraffin-embedded human spleen, using TBX21 Antibody.
M00404-1 · IHCImmunohistochemical analysis of paraffin-embedded human spleen, using T-bet/Tbx21 Antibody.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000073861-TBX21/tissue/cerebral+cortex#imid_8489460
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000073861-TBX21/tissue/colon#imid_8488394
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000073861-TBX21/tissue/liver#imid_8488387
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000073861-TBX21/tissue/kidney#imid_8488380
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for TBX21 IHC & IF
🧪Run spleen first and expect nuclear staining in cells of the red pulp (HPA: Medium in spleen red-pulp cells; UniProt: nucleus). Use adipose tissue as the negative tissue, with adipocytes unstained (HPA: Not detected in adipocytes); neighboring cells without nuclear signal on the spleen slide should show counterstain only (standard IHC practice).
Positive control tissue: Bone marrow (Hematopoietic 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 TBX21; derive a cell-line control from the positive tissue's cell type (Hematopoietic cells) and confirm it by RNA or western blot first.
Technical controls: Include no-primary (secondary-only) and host-species- and antibody-format-matched isotype controls, plus a TBX21 knockout biological negative if available (standard IHC practice). Block endogenous peroxidase and assess pigment in spleen red pulp before interpreting chromogenic signal (standard IHC practice).
⚠️Feasibility: No target-specific fixation window or antigen-retrieval dependence is reported in the supplied evidence; optimize retrieval on paraffin sections (standard IHC practice). The M00404 paraffin-spleen caption leaves the fixative unreported (caption: fixative not stated), and the evidence does not establish whether frozen sections or IF/ICC would be easier (HPA: no cell lines with ICC-IF images). Spleen red-pulp pigment and endogenous peroxidase can complicate chromogenic interpretation (standard IHC practice).
HPA tissue IHC evidence for TBX21
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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 |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Kidney | Proximal tubules (cell body) | Medium | Protein (IHC) | HPA → |
| Lymph node | Non-germinal center cells | Medium | Protein (IHC) | HPA → |
| Spleen | Cells in red pulp | Medium | Protein (IHC) | HPA → |
| Tonsil | Non-germinal center 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 → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced TBX21 IHC Tips
Troubleshoot TBX21 chromogenic IHC in paraffin sections by checking nuclear staining, tissue controls, processing, and detection before interpreting cell counts.
How should I adjust retrieval when TBX21 nuclear staining is weak?
Start with Tris-EDTA pH 9.0 heat-induced epitope retrieval at 95–98 °C for 20 min (page retrieval rule: nuclear antigen). Cool sections gradually, then compare nuclear signal and tissue damage across adjacent sections processed together (standard IHC practice). If staining remains weak, test a shorter or longer heating interval while holding buffer and detection conditions constant (standard IHC practice). Use paraffin-embedded human spleen as a reference tissue because the selected catalog antibody has a tissue-IHC image there; its caption does not report the fixative (catalog antibody M00404 caption). Score signal in individual immune-cell nuclei rather than overall section darkness (HPA: selective nuclear expression in a subset of immune cells).
Could fixation explain inconsistent TBX21 staining across paraffin blocks?
Target-specific TBX21 sensitivity to fixation is unknown from the supplied evidence; the selected spleen caption reports paraffin embedding but no fixative (catalog antibody M00404 caption). Record the fixative, fixation duration, and processing history for each block before comparing results (standard IHC practice). Compare adjacent sections with the same retrieval, IHC-validated antibody dilution, detection reagents, and development time so processing differences are easier to identify (standard IHC practice). If nuclear signal varies between blocks, repeat staining with a shared reference section in each run and inspect morphology for processing damage (standard IHC practice). Do not assign the variation to a particular fixation mechanism without a controlled comparison (standard IHC practice).
What localisation should count as credible TBX21 staining?
Prioritise discrete nuclear chromogen in a subset of immune cells because TBX21 is nuclear and HPA reports selective nuclear expression in that population (UniProt Q9UL17 subcellular; HPA: tissue IHC profile). Evaluate each nucleus against its adjacent cytoplasm and the local background rather than calling diffuse brown tissue positive (standard IHC practice). Spleen red-pulp cells and lymph-node non-germinal-center cells provide reported staining contexts for comparison (HPA: spleen and lymph-node tissue IHC). Diffuse cytoplasmic signal alone does not match the expected compartment and should prompt a check of antibody concentration, detection background, and counterstain (UniProt Q9UL17 subcellular; standard IHC practice). Keep cell identity provisional when morphology alone cannot distinguish neighboring immune-cell populations (standard IHC practice).
How can epitope uncertainty affect TBX21 nuclear IHC?
The supplied record lists a single 1–535 chain and 0 annotated isoforms, but it does not specify the catalog antibody's epitope (UniProt Q9UL17 processing and isoforms; catalog antibody M00404 caption). TBX21 has reported modified residues, including phosphorylation at Ser-53, Tyr-77, Tyr-118, Tyr-220, and Ser-225 (UniProt Q9UL17 modified residues). Without an epitope map, do not claim that a particular modification masks this antibody's binding site (UniProt Q9UL17 modified residues; standard IHC interpretation). If two antibodies give discordant nuclear patterns, compare their documented epitopes, retrieval conditions, and staining of matched sections before assigning biological differences (standard IHC practice). Treat staining with this antibody as TBX21 immunoreactivity, not an isoform- or phosphorylation-specific readout (catalog antibody M00404 caption; UniProt Q9UL17 isoforms).
How should I adapt the interpretation for multiplex TBX21 IF?
For multiplex IF, pair nuclear TBX21 staining with a validated marker of the immune-cell population being assessed, then check colocalisation cell by cell (UniProt Q9UL17 subcellular; HPA: selective nuclear expression in immune cells; standard IF practice). Choose a fluorophore channel after measuring tissue autofluorescence and run single-color controls to assess bleed-through (standard IF practice). TBX21 has no transmembrane segment and is nuclear, so use a permeabilisation condition that permits antibody access through the cell and nuclear boundaries (UniProt Q9UL17 topology and subcellular; standard IF practice). Optimise permeabilisation against morphology and marker retention on matched samples rather than assuming the chromogenic IHC conditions transfer directly (standard IF practice). The selected catalog caption documents paraffin-section IHC, without reporting an IF validation or fixative (catalog antibody M00404 caption).
What should I check when TBX21 chromogen appears outside nuclei?
First inspect no-primary and reagent-only controls for detection-system staining, and compare them with the suspect section (standard IHC practice). Quench endogenous peroxidase before an HRP-based chromogen step and keep DAB development matched between sections; these are general chromogenic IHC controls (standard IHC practice). Recheck blocking, washing, antibody dilution, and section drying if diffuse cytoplasmic color or staining along tissue edges persists (standard IHC practice). True TBX21 staining should be assessed as selective nuclear signal, so broad extracellular or uniformly cytoplasmic color needs independent confirmation (UniProt Q9UL17 subcellular; HPA: tissue IHC profile). Include the same reference tissue in each run to separate batch background from sample-specific effects (catalog antibody M00404 caption; standard IHC practice).
How should I quantify TBX21-positive cells in chromogenic sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define a positive cell by nuclear chromogen above a threshold set with matched control sections, then keep that threshold fixed across the comparison (UniProt Q9UL17 subcellular; standard IHC practice). Report the percentage of positive nuclei among the relevant viable cells, or positive-cell density per mm² of annotated tissue (standard IHC practice). An H-score can combine nuclear intensity with the fraction of cells at each intensity level when intensity is reproducible (standard IHC practice). Normalise counts to the same tissue area or clearly defined cell population, excluding folds, necrosis, and empty space (standard IHC practice). Document regions sampled because HPA describes selective expression in only a subset of immune cells (HPA: tissue IHC profile).
How can I distinguish true TBX21 signal from staining artefacts?
A credible result shows discrete nuclear staining in an appropriate subset of immune cells, consistent with TBX21's nuclear location and HPA's selective tissue pattern (UniProt Q9UL17 subcellular; HPA: tissue IHC profile). Check a reported positive context, such as spleen red pulp, alongside a no-primary control before interpreting an isolated positive cell (HPA: spleen tissue IHC; standard IHC practice). Treat cytoplasmic-only staining, strong section-edge gradients, and color concentrated in necrotic regions as potential artefacts requiring review (UniProt Q9UL17 subcellular; standard IHC practice). Endogenous peroxidase can mimic chromogen signal, so assess the peroxidase-block control when using HRP and DAB (standard IHC practice). Nuclear immunoreactivity identifies TBX21 antigen under these staining conditions; it does not establish transcriptional activity by itself (UniProt Q9UL17 function; standard IHC interpretation).
Boster reagents
Best TBX21 / T-box transcription factor TBX21 IHC Antibodies
Two human-reactive anti-TBX21 antibodies have IHC images from paraffin-embedded human spleen (catalog: M00404 and M00404-1 image captions).
Real IHC data
Anti-TBX21 Rabbit Monoclonal Antibody
Cat # M00404
Real IHC data
Anti-TBX21 Rabbit Monoclonal Antibody
Cat # M00404-1
M00404 is listed for human IHC and Flow Cytometry, with an IHC image from paraffin-embedded human spleen (catalog: applications, reactivity, M00404 image caption). M00404-1 is listed for human IHC, with its own image from paraffin-embedded human spleen (catalog: applications, reactivity, M00404-1 image caption).
Which to pick: For human paraffin-section IHC, both rabbit monoclonals have supporting images; M00404 lists 1:50–1:200 (catalog: clone AFHO-20, dilution; M00404 image caption), while M00404-1 lists 1:50 (catalog: clone 20T77, dilution; M00404-1 image caption). Neither caption reports the fixative (catalog: M00404 and M00404-1 image captions). Neither SKU lists IF/ICC or species beyond human, so the payload supports no IF/ICC or cross-species pick (catalog: applications, reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9UL17 (TBX21_HUMAN, T-box transcription factor TBX21).
- Human Protein Atlas. TBX21 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. TBX21 subcellular location (ICC-IF): Highest expression in HDLM-2: 12.1 nTPM.
- Human Protein Atlas. TBX21 antibody validation summary (2 antibodies).
- Clinicopathological implications of immunohistochemical expression of TBX21, CXCR3, GATA3, CCR4, and TCF1 in nodal follicular helper T-cell lymphoma and peripheral T-cell lymphoma, not otherwise specified. Journal of pathology and translational medicine 2024 — PMC10948251.
- Expression of telomerase reverse transcriptase in peripheral T-cell lymphoma. Cancer medicine 2021 — PMC8495278.
- TBX21 correlates with an immunosuppressive tumor microenvironment and Treg/Th17 imbalance in prostate cancer. Frontiers in oncology 2025 — PMC12819308.
- TBX21 predicts prognosis of patients and drives cancer stem cell maintenance via the TBX21-IL-4 pathway in lung adenocarcinoma. Stem cell research & therapy 2018 — PMC5883886.
- PubMed PMID:10761931 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:15806396 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for TBX21 (T-box transcription factor TBX21). Expected localisation Nuclear staining in a subset of immune cells (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: M00404 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Bone marrow (Hematopoietic cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human spleen tissue; fixative not specified (datasheet M00404); 2. Fixation: Image fixative and duration unreported (datasheet M00404); 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 AFHO-20) anti-TBX21, 1:50-1:200 (datasheet M00404); 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 M00404); Caveat: Kidney proximal tubules also show medium staining (HPA tissue IHC); Isoform / epitope: No annotated isoforms; one 1–535 chain (UniProt); Regulation: T-cell-specific expression (UniProt). Evidence: UniProt Q9UL17, HPA tissue IHC · Enhanced, HPA cell-line RNA · Highest expression in HDLM-2: 12.1 nTPM, Datasheet M00404, PMC10948251. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/TBX21/immunohistochemistry-ihc-guide.