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TNFRSF21 / Tumor necrosis factor receptor superfamily member 21 · IHC design guide
Design Immunohistochemistry for TNFRSF21
Plan TNFRSF21 paraffin IHC using CNS tissue as a strong-staining reference (HPA tissue IHC). Interpret cytoplasmic staining alongside the receptor’s membrane location and ROS-linked endocytosis (HPA tissue IHC; UniProt).
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); cell membrane location (UniProt) | |
| Staining pattern | CNS neuronal cells show strong cytoplasmic staining (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 | 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-linked endocytosis may reduce surface staining (UniProt) | |
| Regulation | Higher in Alzheimer disease brain (UniProt) | |
| Isoform / epitope | No isoforms listed; epitope side of the membrane matters (UniProt) |
Section 1
Recommended TNFRSF21 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet A04348-2) with a published TNFRSF21 IHC protocol using pretreatment breast cancer biopsies (PMC12027272).
| 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 retrieval at pH 8.0 for the catalog antibody (datasheet A04348-2). The published protocol used microwave citrate retrieval at pH 6.0 with a different antibody (PMC12027272).
Section 2
What Is the Expected TNFRSF21 Staining Pattern?
TNFRSF21 (DR6) is a single-pass cell-membrane receptor with an extracellular region and cytoplasmic tail (UniProt O75509 topology). In tissue IHC, expect predominantly cytoplasmic staining in several tissues, strongest in CNS cells: cerebellar molecular-layer cells and neurons of the cerebral cortex and hippocampus (HPA: tissue IHC). Interpret that pattern with the reported medium consistency between antibody staining and RNA expression (HPA: Approved reliability).
| Strong staining in cerebellar molecular-layer cells or cortical and hippocampal neurons, mainly cytoplasmic (HPA: High; tissue IHC). | This matches the reported CNS pattern (HPA: tissue IHC). Some membrane-associated signal is biologically plausible because TNFRSF21 is a membrane receptor (UniProt O75509 topology); HPA tissue IHC does not require a crisp membrane outline. |
| Predominantly nuclear staining, especially without the expected CNS-cell pattern. | Treat an isolated nuclear pattern as suspect: the reported locations are cell membrane (UniProt O75509 subcellular) and cytoplasm in tissue IHC (HPA: tissue IHC). Check controls and antibody-specific documentation before assigning it to TNFRSF21. |
| Strong staining of bone-marrow hematopoietic cells instead of the expected CNS cells (HPA: bone marrow Not detected; CNS High). | Consider cross-reactivity or endogenous chromogenic activity. The HPA observation is a staining comparison, not proof that every hematopoietic cell lacks TNFRSF21 (HPA: tissue IHC). Compare with a no-primary control. |
| Diffuse color across cells, stroma, and slide background, with little separation between structures. | This does not reproduce HPA's cell-associated tissue pattern (HPA: tissue IHC). In chromogenic IHC, nonspecific antibody binding, incomplete blocking, or detection-system background are general possibilities; inspect a no-primary control. |
| No detectable stain in cerebellar molecular-layer cells or cortical and hippocampal neurons (HPA: High in each listed cell population). | A negative result in these reference tissues is inconclusive until tissue quality, primary-antibody performance, retrieval, and chromogenic detection are checked. HPA's Approved rating indicates medium consistency, not guaranteed staining in every preparation (HPA: reliability). |
💡Expected TNFRSF21 appearanceCall positive when CNS target cells show clear, mainly cytoplasmic signal above surrounding background, strongest in cerebellar molecular-layer cells or cortical and hippocampal neurons (HPA: tissue IHC); isolated nuclear staining or uniform color outside cells is suspect against the reported membrane/cytoplasmic locations (UniProt O75509; HPA: tissue IHC).
| Membrane topology and tissue appearance (UniProt O75509 topology; HPA: tissue IHC). | The extracellular region spans residues 42–349, the membrane segment 350–370, and the cytoplasmic region 371–655 (UniProt O75509 topology). HPA nevertheless reports predominantly cytoplasmic tissue staining; do not require a membrane-only IHC pattern (HPA: tissue IHC). |
| Condition-dependent internalization (UniProt O75509 subcellular). | UniProt reports endocytosis following oxidation in response to ROS. Intracellular signal can therefore be compatible with receptor biology, but an ordinary tissue section does not establish that ROS-driven internalization occurred (UniProt O75509 subcellular). |
| Choice of reference tissue (HPA: tissue IHC). | HPA reports High staining in the listed CNS cell populations, Medium in several other listed tissues, and Not detected in bone-marrow hematopoietic cells. Compare the same cell types and interpret intensity relatively; these observations are not universal positive or negative guarantees (HPA: tissue IHC). |
| Antibody evidence (HPA: antibody validation; tissue reliability). | HPA006746 and CAB009805 are each Approved for IHC; the tissue profile carries medium consistency between antibody staining and RNA expression (HPA: antibody validation; tissue reliability). These ratings support comparison with the reported pattern, while leaving individual results open to control-based review. |
| IF/ICC Q&A: Where should fluorescence appear? (HPA: subcellular ICC-IF). | HPA reports plasma membrane (supported) and cytosol (approved), with images from U-251MG and U2OS (HPA: subcellular ICC-IF). That supports a location check for IF/ICC; its staining conditions belong to the separate IF/ICC guide. |
| Situation | Likely cause | Next action |
|---|---|---|
| Weak or absent CNS-cell signal in a reference section (HPA: CNS High). | The staining run may have failed, or the selected antibody and preparation may not reproduce the reported pattern; HPA gives medium consistency (HPA: reliability). | Confirm the expected cells are present, review the antibody's IHC-P instructions, and check retrieval and detection against a working positive control (general IHC practice). |
| Strong staining confined to nuclei, with little cytoplasmic signal. | An isolated nuclear pattern disagrees with membrane localization (UniProt O75509) and the reported cytoplasmic tissue profile (HPA: tissue IHC); specificity is uncertain. | Check a no-primary control, review antibody-specific validation, and compare with CNS-cell staining in the same run (general IHC practice; HPA: CNS High). |
| Color appears broadly in bone-marrow hematopoietic cells (HPA: Not detected). | Cross-reactivity or endogenous chromogenic activity is possible; the HPA observation alone cannot identify which cause applies (HPA: tissue IHC). | Run a no-primary detection control and review endogenous-enzyme blocking appropriate to the chromogen system (general chromogenic IHC practice). |
| Background obscures cell boundaries throughout the section. | Nonspecific binding or detection-system background may overwhelm the cell-associated pattern described by HPA (HPA: tissue IHC). | Compare a no-primary control; review blocking, primary-antibody dilution, washing, and detection timing for the selected IHC workflow (general IHC practice). |
| An otherwise positive section lacks a crisp membrane rim. | That alone is not a failure: HPA describes cytoplasmic tissue staining, although UniProt places the receptor at the cell membrane (HPA: tissue IHC; UniProt O75509). | Score the reported target cells and cytoplasmic signal against background; use the separate IF/ICC evidence when the question specifically concerns subcellular location (HPA: tissue IHC; subcellular ICC-IF). |
| A low-staining tissue gives a negative result beside a positive CNS control (HPA: tissue IHC). | HPA lists Low staining in several cell populations, including cardiomyocytes and pancreatic exocrine glandular cells; weak signal may fall below the assay's visual threshold (HPA: tissue IHC). | Record the cell type and comparator result, and avoid calling the low-staining tissue a definitive TNFRSF21-negative control (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: neuronal cells should stain strongly (HPA: High in cerebral cortex neuronal cells). Use bone marrow hematopoietic cells as the negative tissue (HPA: Not detected); non-neuronal cells on the cortex slide may serve as internal background references, but their TNFRSF21 status is not established by the supplied HPA rows.
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) slide, a host- and immunoglobulin-matched rabbit IgG isotype control, and TNFRSF21-knockout tissue as a biological negative (selected A04348-2 IHC caption: rabbit primary; standard IHC practice). Check endogenous peroxidase and biotin with the caption’s biotin–SABC/DAB detection; if imaging cortex by IF, check brain autofluorescence (selected A04348-2 IHC caption; standard IHC/IF practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected A04348-2 tissue-IHC caption does not state the fixative (selected A04348-2 IHC caption). That caption uses heat retrieval in EDTA at pH 8.0 on a paraffin section, but it does not establish that retrieval is required (selected A04348-2 IHC caption). Whether frozen sections or IF are easier is unreported; brain autofluorescence is a practical IF concern (standard IF 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 chromogenic IHC by checking retrieval, compartment, tissue context and controls before interpreting staining intensity.
Which retrieval condition should I start with for TNFRSF21 in paraffin sections?
Start with heat-mediated retrieval in EDTA pH 8.0 for paraffin sections (datasheet A04348-2). That condition accompanies TNFRSF21 staining in a paraffin-embedded human gastric cancer section, but the caption does not state the fixative (datasheet A04348-2). After retrieval, compare serial sections processed together and incubated with 2 μg/mL primary antibody overnight at 4°C (datasheet A04348-2; standard IHC practice). If staining is weak, adjust heating duration or try another retrieval buffer as a documented fallback, while holding detection conditions constant (standard IHC practice). Excessive heating can damage morphology, so inspect tissue preservation alongside staining intensity (standard IHC practice).
How should I troubleshoot TNFRSF21 staining across differently fixed specimens?
The selected paraffin-section caption does not report its fixative, so TNFRSF21-specific fixation sensitivity is unknown (datasheet A04348-2). Record fixative, fixation duration and tissue processing for each specimen before comparing chromogenic signal (standard IHC practice). For a controlled comparison, cut matched sections, use EDTA pH 8.0 heat retrieval and incubate with 2 μg/mL antibody overnight at 4°C (datasheet A04348-2; standard IHC practice). Include a positive-control section in each run to distinguish a processing difference from a failed staining run (standard IHC practice). Evaluate preserved cellular detail and nonspecific staining before attributing a signal difference to fixation (standard IHC practice).
Should TNFRSF21 staining be membranous or cytoplasmic in IHC?
TNFRSF21 is a membrane receptor with an extracellular segment at residues 42–349 and a cytoplasmic segment at 371–655 (UniProt O75509 topology). It can be endocytosed after oxidation in response to reactive oxygen species, so intracellular staining can be biologically plausible (UniProt O75509 subcellular location). HPA reports cytoplasmic staining across several tissues and supports plasma-membrane localisation alongside approved cytosolic localisation (HPA tissue IHC; HPA subcellular). Assess staining against cell boundaries and cytoplasm on well-preserved sections, using a matched negative control to judge diffuse deposit (standard IHC practice). Treat a compartment shift as an observation requiring confirmation, rather than proof of receptor activation (standard IHC practice).
How does epitope position affect TNFRSF21 IHC interpretation?
Check the antibody's documented immunogen or epitope before interpreting a negative section; its position is not supplied in this payload (standard IHC practice). 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). Six annotated glycosylation sites lie in the extracellular region, which can make epitope accessibility a relevant question without establishing an effect for this antibody (UniProt O75509 glycosylation; standard IHC practice). No isoforms are listed in the supplied record, so do not assign differing staining patterns to a named isoform (UniProt O75509 isoform record). Compare retrieval and antibody controls before drawing an epitope-based conclusion (standard IHC practice).
How can I examine TNFRSF21 by IF alongside chromogenic IHC?
Treat IF/ICC as a separate assay and validate its fixation, retrieval and antibody conditions independently of this IHC caption (standard IF practice; datasheet A04348-2). Multiplex TNFRSF21 with a neuronal marker when examining cerebral cortex or hippocampus, where HPA reports high staining in neuronal cells (HPA tissue IHC). Choose fluorophores after inspecting unstained tissue autofluorescence, and include single-stain controls for channel separation (standard IF practice). If the antibody epitope is cytoplasmic, assess permeabilisation; if it is extracellular, compare staining with and without permeabilisation because the epitope position is unspecified here (UniProt O75509 topology; standard IF practice). Compare subcellular patterns cautiously with HPA's plasma-membrane and cytosolic localisation (HPA subcellular).
What should I check when TNFRSF21 IHC shows diffuse brown background?
Inspect a no-primary control and the section edges before changing antibody concentration; diffuse deposit can reflect detection or tissue background (standard IHC practice). The selected caption used 10% goat serum blocking, 2 μg/mL primary antibody overnight at 4°C, and biotin-based DAB detection (datasheet A04348-2). With a peroxidase-based chromogen, include a peroxidase block and check the no-primary section for endogenous enzyme signal (standard IHC practice). Because the caption uses a biotin-based detection system, assess nonspecific detection-system staining with an appropriate control (datasheet A04348-2; standard IHC practice). Preserve the same retrieval and development conditions across control and test sections when judging whether background has improved (standard IHC practice).
How should I score TNFRSF21 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because TNFRSF21 can appear at the plasma membrane and in the cytosol (HPA subcellular; standard IHC practice). Use an H-score based on intensity and percentage positive, or report percentage positive cells within an explicitly defined region (standard IHC practice). For spatial comparisons, count positive cells per mm² and normalise to the number of eligible cells or analysed tissue area, as appropriate (standard IHC practice). Keep retrieval, primary incubation and DAB development consistent across slides; the selected caption used EDTA pH 8.0 retrieval and 2 μg/mL primary antibody (datasheet A04348-2; standard IHC practice). Exclude damaged or necrotic regions using a prespecified rule (standard IHC practice).
How can I distinguish genuine TNFRSF21 staining from artefact?
Compare signal with an expected cell population: HPA reports high staining in cerebral-cortex and hippocampal neuronal cells, but no detected staining in bone-marrow hematopoietic cells (HPA tissue IHC). Check whether convincing cellular signal occupies membrane or cytoplasm, both supported locations, rather than appearing solely as acellular deposits (HPA subcellular; standard IHC practice). Review section edges and necrotic areas separately because staining there can be misleading (standard IHC practice). A no-primary control helps identify detection background, including endogenous peroxidase signal in chromogenic IHC (standard IHC practice). HPA rates tissue staining as Approved with medium consistency against RNA expression, so corroborate unexpected patterns with independent evidence (HPA tissue IHC; standard IHC practice).
Boster reagents
Best TNFRSF21 / Tumor necrosis factor receptor superfamily member 21 IHC Antibodies
One anti-TNFRSF21 antibody has real IHC data from paraffin sections of human gastric, bladder and renal tumors (catalog image captions). Human, mouse and rat reactivity is listed (catalog reactivity).
Real IHC data
Anti-DR6/TNFRSF21 Antibody ®
Cat # A04348-2
A04348-2 will render with a human gastric cancer paraffin-section IHC figure (A04348-2 image caption). Its other captions show human bladder cancer and renal carcinoma paraffin sections; its listed applications include IHC, with human, mouse and rat reactivity (A04348-2 image captions; catalog applications and reactivity).
Which to pick: Choose A04348-2 for paraffin-section tissue IHC: its caption documents EDTA retrieval at pH 8.0 and primary antibody at 2 μg/ml; the fixative is unreported (A04348-2 image caption). No SKU in the payload has listed IF/ICC validation or an IF image, so there is no supported IF/ICC pick (catalog applications and IF image alts). A04348-2 lists mouse and rat reactivity, but its supplied IHC captions show human tissue only; cross-species tissue IHC needs separate validation (catalog reactivity; A04348-2 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); cell membrane location (UniProt). 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: 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-linked endocytosis may reduce surface staining (UniProt); Isoform / epitope: No isoforms listed; epitope side of the membrane 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. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/TNFRSF21/immunohistochemistry-ihc-guide.