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TNFRSF21 / Tumor necrosis factor receptor superfamily member 21 · IHC design guide
Design Immunohistochemistry for TNFRSF21
Plan TNFRSF21 paraffin-section IHC around cytoplasmic staining in CNS tissue (HPA tissue IHC). Use the catalog antibody’s IHC workflow (datasheet A04348-2) and assess staining by cell type and compartment.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
TNFRSF21 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 tissue staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in cortical and hippocampal neurons (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A04348-2) | |
| Positive control | Cerebellum+4 more · see all | |
| Negative control | Bone marrow |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A04348-2) | |
| Caveat | ROS-triggered endocytosis may shift surface signal inward (UniProt) | |
| Regulation | Higher in Alzheimer disease brain (UniProt) | |
| Isoform / epitope | No isoforms; extracellular vs cytoplasmic epitope matters (UniProt) |
Section 1
Recommended TNFRSF21 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet A04348-2) with two published TNFRSF21 IHC protocols (PMC12027272; PMC9962795).
| Sample | Paraffin-embedded human gastric cancer tissue; fixative not specified (datasheet A04348-2) |
| Fixation | Image fixative and duration unreported (datasheet A04348-2); 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 A04348-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A04348-2) |
| Primary antibody | Rabbit anti-TNFRSF21, 2μg/ml (datasheet A04348-2) |
| Primary incubation | Overnight at 4 °C (datasheet A04348-2) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A04348-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | TNFRSF21-positive staining in cells in molecular layer of cerebellum (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several tissues, most abundant in CNS. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA, pH 8.0, for the catalog antibody (datasheet A04348-2). The breast biopsy protocol instead used microwave-heated citrate, pH 6.0 (PMC12027272).
Section 2
What Is the Expected TNFRSF21 Staining Pattern?
TNFRSF21 is a cell membrane receptor with an extracellular region at residues 42–349 and a cytoplasmic region at 371–655 (UniProt O75509 topology). In paraffin tissue IHC, expect predominantly cytoplasmic staining, strongest in selected CNS cells, including cortical and hippocampal neurons (HPA tissue IHC). HPA rates its tissue staining “Approved,” with medium consistency between staining and RNA expression (HPA tissue IHC reliability).
| Strong staining in cortical or hippocampal neurons, or cells of the cerebellar molecular layer (HPA tissue IHC). | This matches HPA’s High staining in these cell populations; a predominantly cytoplasmic appearance is consistent with its tissue profile (HPA tissue IHC). Compare the cell population and compartment, since intensity alone is insufficient to judge a slide (general IHC practice). |
| A cell-edge signal accompanies cytoplasmic staining in an otherwise plausible cell population. | A membrane component is biologically plausible because TNFRSF21 spans the membrane (UniProt O75509 topology); HPA also reports plasma membrane and cytosol localisation by ICC-IF (HPA subcellular). The tissue IHC profile is predominantly cytoplasmic, so membrane-only staining needs comparison with tissue controls (HPA tissue IHC; general IHC practice). |
| Nuclear staining dominates, with little staining at the cell edge or in the cytoplasm. | This does not match UniProt’s membrane topology or HPA’s plasma membrane and cytosol localisation (UniProt O75509 topology; HPA subcellular). Treat it as an unconfirmed pattern and check the counterstain, detection controls and antibody conditions before assigning it to TNFRSF21 (general IHC practice). |
| Strong staining appears throughout bone marrow hematopoietic cells. | HPA reports these cells as Not detected (HPA tissue IHC). Widespread strong signal there raises concern about cross-reactivity or endogenous detection activity, though this comparison alone cannot identify the cause (HPA tissue IHC; general IHC practice). |
| Diffuse color covers cells and surrounding tissue, obscuring cell boundaries. | The slide cannot support a compartment or cell-type call while background masks morphology (general IHC practice). Compare a no-primary control and review blocking, washing and detection conditions before interpreting the apparent signal (general IHC practice). |
💡Expected TNFRSF21 appearanceCall a convincing positive when cortical or hippocampal neurons show High, predominantly cytoplasmic staining (HPA tissue IHC); strong nuclear staining or broad signal in bone marrow hematopoietic cells is suspect (UniProt O75509 topology; HPA subcellular; HPA tissue IHC).
| Membrane topology and unknown antibody epitope | TNFRSF21 has extracellular residues 42–349, a transmembrane segment at 350–370 and cytoplasmic residues 371–655 (UniProt O75509 topology). The supplied antibody information gives no epitope, so topology cannot predict which compartment this IHC antibody will emphasize. |
| Oxidation-associated internalisation | UniProt reports endocytosis following oxidation in response to reactive oxygen species (UniProt O75509 subcellular). Intracellular staining is therefore biologically plausible, but a tissue stain alone cannot establish that oxidation or endocytosis occurred in that specimen. |
| Protein processing and glycosylation | UniProt lists a 1–41 signal peptide, a 42–655 chain and six glycosylation sites (UniProt O75509 processing; glycosylation). The payload does not identify the antibody epitope or show that these features alter staining under a particular retrieval condition. |
| Tissue reference and validation limit | HPA reports High staining in selected CNS cells, Medium staining in several other listed cell populations, and no detection in bone marrow hematopoietic cells (HPA tissue IHC). Its “Approved” tissue assessment has medium staining-to-RNA consistency; these observations guide comparison but do not guarantee a result in every section (HPA tissue IHC reliability). |
| Situation | Likely cause | Next action |
|---|---|---|
| No stain in cortical or hippocampal neurons. | These are High reference populations, so a blank slide may reflect assay failure or unsuitable conditions; the image alone cannot establish which (HPA tissue IHC; general IHC practice). | Confirm the control section contains the relevant neurons, then check primary antibody application, retrieval, detection reagents and counterstain visibility (general IHC practice). The supplied sources give no TNFRSF21-specific retrieval setting. |
| Only faint staining in an otherwise intact section. | Some listed populations are Low, including pancreatic exocrine cells and cardiomyocytes, so faint staining there can fit the reference pattern (HPA tissue IHC). | Identify the cell population before changing conditions; compare it with a CNS positive control processed in the same run (HPA tissue IHC; general IHC practice). |
| Strong signal persists in a no-primary control. | That pattern points toward endogenous detection activity or background from detection reagents rather than primary antibody binding (general IHC practice). | Review the detection system’s appropriate endogenous-activity block and the blocking and washing steps, then repeat the control (general IHC practice). |
| Staining is widespread but tissue detail is still visible. | Excessive primary or detection signal, insufficient blocking, or inadequate washing can reduce cell-type contrast (general IHC practice). | Compare no-primary and known-positive controls, then adjust antibody dilution, blocking or washes one variable at a time (general IHC practice). No dilution is supplied for this section. |
| A sharply nuclear pattern is the main result. | Nuclear dominance conflicts with the supplied membrane, cytosol and cytoplasmic localisation evidence (UniProt O75509 topology; HPA subcellular; HPA tissue IHC). | Recheck the counterstain and no-primary control, and compare the same antibody in a CNS positive section before scoring nuclear staining as TNFRSF21 (general IHC practice; HPA tissue IHC). |
| Can ICC-IF show both membrane and cytosolic signal? | Yes. HPA reports supported plasma membrane and approved cytosol localisation, with ICC-IF images in U-251MG and U2OS (HPA subcellular). | Interpret those images as ICC-IF localisation evidence; use the separate IF/ICC guide for its workflow, while using HPA tissue IHC to judge paraffin tissue staining (HPA subcellular; HPA tissue IHC). |
Experiment evidence
IHC & IF images showing TNFRSF21 staining
A04348-2 · IHCIHC analysis of DR6/TNFRSF21 using anti-DR6/TNFRSF21 antibody (A04348-2). DR6/TNFRSF21 was detected in paraffin-embedded section of human ga
A04348-2 · IHCIHC analysis of DR6/TNFRSF21 using anti-DR6/TNFRSF21 antibody (A04348-2). DR6/TNFRSF21 was detected in paraffin-embedded section of human bl
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000146072-TNFRSF21/tissue/cerebral+cortex#imid_2465472
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000146072-TNFRSF21/tissue/colon#imid_2465414
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000146072-TNFRSF21/tissue/liver#imid_2465407
HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000146072-TNFRSF21/tissue/pancreas#imid_2465350
HPA · ICC-IF U-251MGHuman Protein Atlas immunofluorescence, U-251MG, antibody CAB009805; green = TNFRSF21, blue = nucleus, red = microtubules.
HPA · ICC-IF U-251MGHuman Protein Atlas immunofluorescence, U-251MG, antibody CAB009805; green = TNFRSF21, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for TNFRSF21 IHC & IF
🧪Run cerebral cortex first and assess staining in neuronal cells, which HPA rates High (HPA: cerebral cortex, neuronal cells, High). Run bone marrow as the negative tissue and assess hematopoietic cells, which HPA rates Not detected (HPA: bone marrow, hematopoietic cells, Not detected). On the cortex slide, use cells without visible signal as internal background comparators; their identity as true negatives requires separate validation (HPA: High is assigned to neuronal cells).
Positive control tissue: Cerebellum (Cells in molecular layer, HPA High)
Negative control tissue: Bone marrow (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show TNFRSF21 in U-251MG, U2OS, with annotated localisation: Plasma membrane (supported), Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a concentration-matched rabbit isotype control; use TNFRSF21 knockout material or a validated immunizing-peptide block as a biological specificity control (selected-SKU IHC caption: rabbit primary antibody; standard IHC practice). For cortical DAB staining, block endogenous peroxidase and check endogenous biotin background because the selected detection method uses a biotinylated secondary and streptavidin–biotin complex (selected-SKU IHC caption: SABC with DAB; standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the exact selected-SKU tissue-IHC caption does not state a fixative (selected-SKU IHC caption: fixative not stated). Heat retrieval in EDTA at pH 8.0 is a documented starting condition for paraffin-section IHC; the evidence does not establish that retrieval is required for every specimen (selected-SKU IHC caption: heat-mediated EDTA retrieval). Comparative ease of frozen-section IHC or IF is unreported; in cortex, distinguish granular neuronal pigment from DAB signal using the control sections (HPA: cerebral cortex neuronal cells, High; standard IHC practice).
HPA tissue IHC evidence for TNFRSF21
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 |
|---|---|---|---|---|
| Cerebellum | Cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Hippocampus | Neuronal cells | High | Protein (IHC) | HPA → |
| Adipose tissue | Adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced TNFRSF21 IHC Tips
Troubleshoot TNFRSF21 staining in paraffin section IHC by checking retrieval, compartment, cell type and controls before interpreting chromogenic signal.
What retrieval should I start with if TNFRSF21 staining is weak?
Start with heat-mediated EDTA retrieval at pH 8.0 for paraffin sections (datasheet A04348-2). The selected image used this retrieval before 10% goat serum blocking and 2 µg/ml primary antibody overnight at 4°C (datasheet A04348-2). If staining remains weak, compare a shorter and longer heating exposure on matched sections while keeping the antibody and detection conditions constant (standard IHC practice). Include a known positive neuronal section and a no-primary control so loss of signal and background can be assessed together (HPA: high staining in cerebral cortex neuronal cells; standard IHC practice). Record retrieval conditions with each slide because differences in heating can affect comparisons (standard IHC practice).
Could fixation explain inconsistent TNFRSF21 staining between paraffin blocks?
TNFRSF21-specific sensitivity to fixation is unknown from the supplied evidence (datasheet A04348-2: fixative not stated). The selected image shows staining in a paraffin-embedded human gastric cancer section, but its caption does not identify the fixative or fixation duration (datasheet A04348-2). For inconsistent blocks, document their actual fixation histories and process matched sections with the same EDTA pH 8.0 retrieval and detection sequence (datasheet A04348-2; standard IHC practice). Compare a known positive control on each run before attributing a weak block to biology (HPA: high staining in cerebral cortex neuronal cells; standard IHC practice). Avoid assigning a TNFRSF21-specific fixation effect without a controlled comparison (standard IHC practice).
Should TNFRSF21 staining appear at the membrane or in the cytoplasm?
Expect a membrane-associated pattern because TNFRSF21 has an extracellular region at residues 42–349, a transmembrane segment at 350–370, and a cytoplasmic region at 371–655 (UniProt O75509 topology). Cytoplasmic signal can also be biologically plausible: TNFRSF21 is endocytosed after ROS-associated oxidation, and HPA reports cytoplasmic tissue staining (UniProt O75509 subcellular location; HPA: tissue IHC profile). HPA additionally supports plasma membrane localisation and approves cytosolic localisation in cell images (HPA: subcellular localisation). In chromogenic sections, compare both compartments in identifiable cells against adjacent background and a no-primary control (standard IHC practice). Diffuse signal alone cannot establish receptor localisation (standard IHC practice).
How should epitope location change my reading of TNFRSF21 staining?
First check the antibody's stated immunogen or epitope before assigning a particular TNFRSF21 domain to its staining; the supplied caption does not specify one (datasheet A04348-2). TNFRSF21 has an extracellular region at residues 42–349 and a cytoplasmic region at 371–655, including a death domain at 415–498 (UniProt O75509 topology and domains). Its signal peptide spans 1–41, and 6 glycosylation sites are listed in the extracellular region (UniProt O75509 processing and glycosylation). An antibody's epitope could therefore affect which processed or accessible receptor pool it reveals, but that cannot be assigned for this catalog antibody from the caption (UniProt O75509 topology; datasheet A04348-2). No isoforms are listed in the supplied record, so do not interpret differing patterns as isoform-specific staining without separate evidence (UniProt O75509 isoforms).
How can I compare TNFRSF21 immunofluorescence with this IHC result?
Treat IF as a separate validation experiment while using the chromogenic IHC pattern as a reference for cell type and compartment (standard IHC/IF practice). Multiplex TNFRSF21 with a validated neuronal marker when examining cerebral cortex or hippocampus, where HPA reports high neuronal staining (HPA: cerebral cortex and hippocampus neuronal cells; standard IF practice). Choose fluorophores and acquisition channels after checking tissue autofluorescence with an unstained section, and include single-stain controls for spectral overlap (standard IF practice). The catalog antibody's epitope side is unspecified, so establish it before selecting permeabilisation: an intracellular epitope requires membrane access, whereas an extracellular epitope may be assessed without it (datasheet A04348-2: epitope unspecified; UniProt O75509 topology; standard IF practice). Compare membrane and cytosolic distributions with the IHC result without assuming identical signal intensity across detection methods (HPA: subcellular localisation; standard IHC/IF practice).
How do I distinguish TNFRSF21 signal from chromogenic background?
Run a no-primary control through the same secondary and chromogen steps to reveal detection-system background (standard IHC practice). The selected IHC image used a biotinylated secondary, streptavidin–biotin detection and DAB, so check tissue-associated biotin and endogenous peroxidase contributions with appropriate controls (datasheet A04348-2; standard IHC practice). Block endogenous peroxidase before DAB development as part of the general chromogenic workflow, and compare background after the stated 10% goat serum block (standard IHC practice; datasheet A04348-2). If staining is widespread, compare the 2 µg/ml primary condition with a lower concentration on matched sections while preserving exposure and detection settings (datasheet A04348-2; standard IHC practice). Judge specific signal within cell boundaries against adjacent negative areas rather than from overall slide darkness (standard IHC practice).
What should I quantify when comparing TNFRSF21 across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring: HPA reports high neuronal staining in cerebral cortex and hippocampus and a cytoplasmic tissue profile (HPA: tissue IHC profile). For matched chromogenic sections, report the percentage of positive cells and an H-score on a 0–300 scale, using the same intensity thresholds and cell selection rules throughout (standard IHC practice). Normalise positive counts to the number of eligible cells, or report positive-cell density per mm² of viable tissue when cell counting is impractical (standard IHC practice). Exclude folds, tissue edges and necrotic regions by a prespecified rule, and retain representative images for review (standard IHC practice). Keep retrieval, antibody concentration, development time and imaging settings consistent across compared sections (standard IHC practice).
When is a TNFRSF21-positive area credible rather than artefactual?
A credible signal should occupy identifiable cells and a plausible receptor compartment: TNFRSF21 is a membrane protein that can be endocytosed, while HPA reports cytoplasmic tissue staining (UniProt O75509 topology and subcellular location; HPA: tissue IHC profile). High staining in cerebral cortex or hippocampal neurons provides a documented comparison, whereas HPA reports hematopoietic cells in bone marrow as not detected (HPA: tissue IHC profile). Do not interpret a brown rim at tissue edges, necrotic areas or staining reproduced by a no-primary control as target evidence (standard IHC practice). Check endogenous peroxidase and biotin contributions when using the caption's DAB and streptavidin–biotin workflow (datasheet A04348-2; standard IHC practice). Interpret unexpected cells or compartments cautiously because HPA rates agreement between tissue staining and RNA expression as medium (HPA: Approved, medium consistency).
Boster reagents
Best TNFRSF21 / Tumor necrosis factor receptor superfamily member 21 IHC Antibodies
A04348-2 has IHC images from paraffin-embedded human gastric cancer, bladder cancer, and renal carcinoma sections (catalog image captions). Human, mouse, and rat reactivity is listed (catalog reactivity).
Real IHC data
Anti-DR6/TNFRSF21 Antibody ®
Cat # A04348-2
A04348-2 is listed for IHC and has images from paraffin-embedded human gastric cancer, bladder cancer, and renal carcinoma sections (catalog applications; image captions). It is listed as reactive with human, mouse, and rat, but the supplied IHC images show human tissue only (catalog reactivity; image captions).
Which to pick: Choose A04348-2 for paraffin-section IHC; its human tissue captions report EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody (catalog image captions). No IF/ICC application or image is supplied, so an IF/ICC choice cannot be supported here (catalog applications; image captions). Human, mouse, and rat reactivity is listed for A04348-2, but the supplied IHC images establish only human tissue use; the fixative is unreported (catalog reactivity; image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O75509 (TNR21_HUMAN, Tumor necrosis factor receptor superfamily member 21).
- Human Protein Atlas. TNFRSF21 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. TNFRSF21 subcellular location (ICC-IF): Localized to the plasma membrane and cytosol..
- Human Protein Atlas. TNFRSF21 antibody validation summary (2 antibodies).
- M2 macrophage facilitated angiogenesis in cutaneous squamous cell carcinoma via circ_TNFRSF21/miR-3619-5p/ROCK axis. The Kaohsiung journal of medical sciences 2022 — PMC11896439.
- Integrating Machine Learning and Bulk and Single-Cell RNA Sequencing to Decipher Diverse Cell Death Patterns for Predicting the Prognosis of Neoadjuvant Chemotherapy in Breast Cancer. International journal of molecular sciences 2025 — PMC12027272.
- Comprehensive Analysis of Necroptosis Landscape in Skin Cutaneous Melanoma for Appealing its Implications in Prognosis Estimation and Microenvironment Status. Journal of personalized medicine 2023 — PMC9962795.
- APP binds DR6 to trigger axon pruning and neuron death via distinct caspases. Nature 2009 — PMC2677572.
- PubMed PMID:9714541 — UniProt-cited evidence.
- PubMed PMID:12975309 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for TNFRSF21 (Tumor necrosis factor receptor superfamily member 21). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A04348-2). Validated IHC antibody: A04348-2 at 2μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Cerebellum (Cells in molecular layer); negative Bone marrow. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human gastric cancer tissue; fixative not specified (datasheet A04348-2); 2. Fixation: Image fixative and duration unreported (datasheet A04348-2); 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 A04348-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A04348-2); 8. Primary antibody: Rabbit anti-TNFRSF21, 2μg/ml (datasheet A04348-2); 9. Primary incubation: Overnight at 4 °C (datasheet A04348-2). Watch for: Fixation: Keep fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A04348-2); Caveat: ROS-triggered endocytosis may shift surface signal inward (UniProt); Isoform / epitope: No isoforms; extracellular vs cytoplasmic epitope matters (UniProt); Regulation: Higher in Alzheimer disease brain (UniProt). Evidence: UniProt O75509, HPA tissue IHC · Approved, HPA ICC-IF · plasma membrane and cytosol., Datasheet A04348-2, PMC12027272, PMC9962795. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/TNFRSF21/immunohistochemistry-ihc-guide.