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THBS2 / Thrombospondin-2 · IHC design guide
Design Immunohistochemistry for THBS2
Plan paraffin-section THBS2 IHC using the granular cytoplasmic tissue pattern reported by HPA (HPA tissue IHC). This guide covers fixation, retrieval, controls and interpretation, including the possibility that a secreted protein stains away from its RNA source (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
THBS2 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 | Granular cytoplasmic staining in several tissues (HPA tissue IHC) | |
| Staining pattern | Granular cytoplasm in adipocytes and glandular cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Adipose tissue+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Secreted THBS2 may stain away from its RNA source (HPA tissue IHC) | |
| Regulation | Blood vessel–enhanced RNA expression (HPA tissue RNA) | |
| Isoform / epitope | No annotated isoforms; mature chain starts at residue 19 (UniProt) |
Section 1
Recommended THBS2 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet) with published THBS2 staining protocols for bowel (PMC11178913), pancreatic (PMC5727893), and gastric tissue (PMC4189190).
| Sample | Paraffin-embedded rat testis tissue; fixative not specified (datasheet A03253) |
| Fixation | Image fixative and duration unreported (datasheet A03253); 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 citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane 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 anti-THBS2, 1:100 (datasheet A03253) |
| 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 | THBS2-positive staining in adipocytes of adipose tissue (HPA tissue IHC: Medium). HPA tissue profile: Granular cytoplasmic expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval), then compare the published citrate conditions (PMC5727893; PMC4189190).
Section 2
What Is the Expected THBS2 Staining Pattern?
In paraffin-section IHC, expect granular cytoplasmic THBS2 staining in selected cells, including adipocytes, bronchial respiratory epithelial cells and cerebral cortical neurons (HPA tissue IHC). HPA rates the tissue pattern Approved, with medium consistency against RNA; it cautions that secreted protein and RNA locations can differ (HPA tissue IHC). THBS2 has a signal peptide and no transmembrane segment, while UniProt provides no subcellular location annotation (UniProt P35442).
| Granular cytoplasmic staining of adipocytes, with a moderate signal (HPA tissue IHC). | This matches an HPA-observed positive pattern. Compare the stained cells and signal with a known-positive section processed in the same IHC run; the HPA rating is Approved, with medium consistency against RNA (HPA tissue IHC). |
| Predominantly nuclear chromogen in cells expected to show granular cytoplasmic staining (HPA tissue IHC). | Treat this as a discordant compartment pattern and investigate artefact before calling THBS2 positive. HPA reports granular cytoplasmic tissue staining; its plasma-membrane finding comes from ICC-IF and does not validate a nuclear IHC pattern (HPA tissue IHC; HPA subcellular ICC-IF). |
| Strong staining in germinal-center cells or oral squamous epithelial cells (HPA tissue IHC). | Both are listed as Low by HPA, so strong signal merits checks for antibody cross-reactivity or endogenous detection activity. Low is not a verified negative; do not use either cell population alone to declare a stain nonspecific (HPA tissue IHC). |
| Uniform chromogen across cells and tissue spaces, obscuring cellular detail. | Diffuse background prevents assessment of the HPA granular cytoplasmic pattern (HPA tissue IHC). Check a primary-antibody-omission control for detection background and review blocking, washes and chromogen development (general IHC practice). |
| No detectable staining in an adipose-tissue section chosen as a positive control (HPA tissue IHC). | Adipocytes are reported at Medium staining, so a blank result calls for a run-level check, not an immediate biological conclusion (HPA tissue IHC). Review section quality, antibody compatibility, retrieval and detection controls (general IHC practice). |
💡Expected THBS2 appearanceCall a positive result when selected cells show discernible granular cytoplasmic staining, such as Medium staining in adipocytes; uniform background or dominant nuclear chromogen is discordant (HPA tissue IHC).
| Tissue and cell selection | HPA reports Medium staining in adipocytes, adrenal and breast glandular cells, bronchial respiratory epithelial cells, and several brain cell populations; it lists some other cells as Low, with no verified negatives in this payload (HPA tissue IHC). |
| Secretion and compartment | The signal peptide spans residues 1–18, the mature chain spans 19–1172, and no transmembrane segment is annotated (UniProt P35442). Interpret tissue staining in light of HPA's warning that secreted protein and RNA can occupy different locations (HPA tissue IHC). |
| Antibody evidence | HPA066883 and CAB017716 have IHC Approved status, whereas HPA069350 has no IHC status listed (HPA antibodies). Approved supports the reported observations but does not remove the need for controls when the sample shows a different pattern (HPA tissue IHC; general IHC practice). |
| Epitope and processing | UniProt identifies a cleaved signal peptide and a mature THBS2 chain beginning at residue 19 (UniProt P35442). An antibody's epitope is not supplied here, so these records cannot predict whether its IHC staining depends on a particular region or retrieval condition. |
| Antigen retrieval | Use the chosen IHC-validated antibody's documented paraffin-section conditions and compare retrieval settings with matched control sections (general IHC practice). Neither HPA tissue staining nor UniProt sequence features establish THBS2-specific fixation sensitivity or an optimal retrieval method. |
| IF/ICC Q&A | Q: Should IF/ICC match the tissue IHC compartment? A: HPA ICC-IF reports mainly plasma-membrane staining, with additional cytosol and centrosome localization; that assay-specific observation does not replace the granular cytoplasmic tissue-IHC description (HPA subcellular ICC-IF; HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| The known-positive adipocyte control is blank (HPA tissue IHC). | An IHC run or reagent problem is possible; HPA reports Medium adipocyte staining, but its Approved rating does not guarantee every section will stain (HPA tissue IHC). | Confirm tissue preservation and section identity, then check the antibody's IHC-P instructions, retrieval setup and detection reagents with a concurrent positive control (general IHC practice). |
| Most structures show diffuse brown signal. | Detection background or overdevelopment can obscure the expected granular cytoplasmic pattern (HPA tissue IHC; general IHC practice). | Inspect the primary-antibody-omission control; review blocking, wash stringency and chromogen development before scoring individual cells (general IHC practice). |
| Germinal-center cells stain strongly (HPA tissue IHC). | HPA lists these cells as Low; strong staining could reflect cross-reactivity or endogenous detection activity, but Low does not mean absent (HPA tissue IHC). | Compare a matched omission control and an HPA Medium-positive tissue section; investigate detection background before assigning THBS2 specificity (HPA tissue IHC; general IHC practice). |
| Nuclear staining dominates the slide. | This conflicts with HPA's granular cytoplasmic tissue-IHC profile and should be treated as a possible artefact (HPA tissue IHC). | Recheck the counterstain and chromogen under brightfield, inspect controls, and score THBS2 only when the cellular staining pattern is interpretable (general IHC practice). |
| Tissue RNA and protein locations appear different. | HPA explicitly expects location differences for a secreted protein and rates antibody-staining/RNA consistency as medium (HPA tissue IHC). | Use the stained cell pattern and appropriate IHC controls for interpretation; avoid treating RNA location alone as proof that the protein stain is false (HPA tissue IHC; general IHC practice). |
| An antibody selected from the HPA list gives an uncertain paraffin-section result. | HPA069350 lists ICC Approved but no IHC status; HPA066883 and CAB017716 list IHC Approved (HPA antibodies). | Check the exact antibody identifier and its IHC-P documentation, then interpret only alongside positive and omission controls (HPA antibodies; general IHC practice). |
Experiment evidence
IHC & IF images showing THBS2 staining
A03253 · IHCImmunohistochemical analysis of paraffin-embedded rat-testis, antibody was diluted at 1:100
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000186340-THBS2/tissue/cerebral+cortex#imid_4628856
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000186340-THBS2/tissue/colon#imid_4628798
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000186340-THBS2/tissue/liver#imid_4628799
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000186340-THBS2/tissue/kidney#imid_4628800
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB017716; green = THBS2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB017716; green = THBS2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for THBS2 IHC & IF
🧪Run adipose tissue first and look for staining in adipocytes (HPA: Medium in adipocytes). HPA detects THBS2 in all 45 scored tissues, so use no-primary and isotype controls for the negative; cells without visible signal on the positive slide can help assess background but are not validated THBS2-negative cells (HPA: no negative tissue rows).
Positive control tissue: Adipose tissue (Adipocytes, HPA Medium)
Negative control tissue: None in HPA: THBS2 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show THBS2 in A-431, U-251MG, U2OS, ASC52telo, with annotated localisation: Plasma membrane (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide, an isotype control matched to the primary antibody’s host species and clonality, and THBS2-knockout material as a biological negative. For chromogenic adipose IHC, block endogenous peroxidase and check that empty lipid spaces are not scored as cellular staining (standard IHC practice).
⚠️Feasibility: A THBS2-specific fixation window or fixation effect is unreported in the supplied evidence; the selected A03253 paraffin rat-testis caption gives 1:100 but does not report a fixative (selected tissue-IHC caption). Antigen-retrieval dependence is unreported, so optimize retrieval empirically for IHC-P; the supplied evidence does not establish that frozen sections or IF are easier. Paraffin processing can leave empty lipid spaces in adipose tissue, which complicates interpretation of staining around adipocytes (standard histology practice).
HPA tissue IHC evidence for THBS2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Secreted protein, tissue location of RNA and protein is expected to differ.). 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 |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: THBS2 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced THBS2 IHC Tips
Troubleshoot THBS2 staining by checking retrieval, tissue processing, compartment, controls and scoring before interpreting chromogenic signal.
What retrieval should I start with if THBS2 staining is weak in paraffin sections?
Start with citrate pH 6.0 heat retrieval at 95–98 °C for 20 min (page retrieval rule: cytoplasmic / membrane antigen). If staining remains weak, compare a longer citrate incubation with a small pilot using an alternative retrieval buffer, while keeping antibody concentration and detection conditions constant (standard IHC practice). Record tissue retention and background alongside signal, since harsher heating can damage sections or expose unwanted binding (standard IHC practice). The selected catalog image establishes staining in paraffin-embedded rat testis at 1:100, but its caption does not report a retrieval method (catalog antibody A03253 caption).
How should I investigate inconsistent THBS2 staining across differently fixed specimens?
THBS2-specific sensitivity to fixation is unknown from the supplied evidence, and the selected paraffin-section caption does not state a fixative (catalog antibody A03253 caption). Document each specimen’s fixative, fixation duration and processing history before comparing chromogenic intensity (standard IHC practice). For a controlled pilot, process matched specimens with a consistent fixation schedule, then hold retrieval, antibody dilution and detection settings constant (standard IHC practice). Assess morphology and staining together, and avoid treating a difference between batches as a biological THBS2 change until processing effects have been checked (standard IHC practice).
Where should THBS2 staining appear, and how should I assess a cytoplasmic pattern?
THBS2 has a signal peptide at residues 1–18 and no transmembrane segment, consistent with a secreted protein that may associate with extracellular matrix (UniProt P35442 processing and topology; UniProt P35442 function). HPA tissue IHC reports granular cytoplasmic expression in several tissues, so that pattern alone is not grounds for rejection (HPA tissue IHC profile). In cell imaging, HPA reports mainly plasma membrane localisation with additional cytosol and centrosome signals, which should not be imposed as a required pattern in paraffin tissue (HPA subcellular). Score cellular and extracellular staining separately, with morphology and control sections guiding interpretation (standard IHC practice).
Could an unmapped epitope explain discordant THBS2 staining between antibody preparations?
The supplied record lists 0 isoforms, so an isoform-specific explanation is unsupported here (UniProt P35442 isoforms). THBS2 contains multiple domains, seven listed glycosylation sites and disulfide-linked homotrimers; epitope exposure may therefore depend on what an antibody recognises, but this antibody’s epitope is not supplied (UniProt P35442 domains, glycosylation and subunit; catalog antibody A03253 caption). Compare preparations on adjacent sections using identical retrieval and detection conditions, then review any available immunogen or epitope documentation before assigning a molecular cause (standard IHC practice). Keep a matched negative control to distinguish altered specific staining from a change in background (standard IHC practice).
How can I assess THBS2 by multiplex IF without misreading extracellular signal?
For the separate IF/ICC workflow, pair THBS2 with a validated marker of the expected cell type; adipocytes are one HPA tissue-IHC positive population, although that observation does not validate an IF result (HPA tissue IHC: adipocytes). Choose fluorophores after inspecting unstained tissue autofluorescence, and reserve a spectrally distinct channel for the weaker signal (standard IF practice). Because THBS2 has no transmembrane segment and this antibody’s epitope is unspecified, compare nonpermeabilised staining with a gently permeabilised condition when testing extracellular versus intracellular accessibility (UniProt P35442 topology; catalog antibody A03253 caption; standard IF practice). Include single-colour and secondary-only controls before interpreting overlap (standard IF practice).
What should I check when THBS2 DAB staining is diffuse or widespread?
Run a no-primary control and check whether colour develops in the same structures; this helps identify detection-system background, including endogenous peroxidase activity (standard IHC practice). Apply a peroxidase block before DAB development and use an appropriate protein block, then compare shorter primary incubation or a lower antibody concentration on matched sections (standard IHC practice). Keep any change separate from retrieval adjustments so the source of improvement is clear (standard IHC practice). Because THBS2 can bind fibronectin, laminin and type V collagen, diffuse matrix-associated signal warrants careful comparison with tissue morphology and controls rather than automatic dismissal (UniProt P35442 subunit).
How should I quantify THBS2 IHC when staining spans cells and matrix? ⚠ ANSWER MARKED FOR VERIFICATION
Define the analysis compartment before scoring, because THBS2 can participate in cell-to-matrix interactions and HPA tissue IHC also describes granular cytoplasmic staining (UniProt P35442 function; HPA tissue IHC profile). For cellular staining, record percent positive cells and intensity or calculate an H-score; for extracellular signal, report stained area or optical density per mm² of evaluable tissue (standard IHC practice). Normalise cellular counts to the relevant cell population and matrix measurements to viable tissue area, excluding folds, edges and necrosis by a prespecified rule (standard IHC practice). Keep imaging, colour separation and thresholds consistent across batches (standard IHC practice).
How can I distinguish credible THBS2 positivity from staining artefact?
A credible result should show reproducible staining in morphologically intact regions and exceed matched negative-control signal under the same detection settings (standard IHC practice). Review cellular granules and plausible matrix-associated staining together: HPA reports granular cytoplasmic tissue staining, while THBS2 is an adhesive protein with matrix-binding partners (HPA tissue IHC profile; UniProt P35442 function and subunit). Flag isolated section-edge colour, necrotic debris or signal that persists without primary antibody as potential artefact, including endogenous enzyme activity (standard IHC practice). Do not equate absent staining in one compartment with absent THBS2 expression, since HPA notes that secreted protein and RNA locations can differ (HPA tissue IHC reliability description).
Boster reagents
Best THBS2 / Thrombospondin-2 IHC Antibodies
A03253 has a rat testis paraffin section IHC image (catalog image caption) and listed IF use with human, mouse and rat reactivity (catalog: applications and reactivity).
Real IHC data
Anti-Thrombospondin 2 Antibody
Cat # A03253
A03253 has an IHC image of paraffin-embedded rat testis stained at 1:100 (catalog image caption). IF is listed at 1:50, with human, mouse and rat reactivity; no IF image is supplied (catalog: applications, dilution, reactivity and image records).
Which to pick: For tissue IHC, A03253 has direct image evidence for rat testis paraffin sections (catalog image caption), although IHC is absent from its listed applications (catalog: applications). For IF, A03253 is the listed option at 1:50; its stated reactivity covers human, mouse and rat, while ICC validation is unreported (catalog: applications, dilution and reactivity). The IHC caption does not report the fixative (catalog image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P35442 (TSP2_HUMAN, Thrombospondin-2).
- Human Protein Atlas. THBS2 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. THBS2 subcellular location (ICC-IF): Mainly localized to the plasma membrane. In addition localized to the cytosol and centrosome..
- Human Protein Atlas. THBS2 antibody validation summary (3 antibodies).
- Thrombospondin 2, matrix Gla protein and digital analysis identified distinct fibroblast populations in fibrostenosing Crohn's disease. Scientific reports 2024 — PMC11178913.
- THBS2 is Closely Related to the Poor Prognosis and Immune Cell Infiltration of Gastric Cancer. Frontiers in genetics 2022 — PMC8853287.
- Detection of early pancreatic ductal adenocarcinoma with thrombospondin-2 and CA19-9 blood markers. Science translational medicine 2017 — PMC5727893.
- Down regulation of Thrombospondin2 predicts poor prognosis in patients with gastric cancer. Molecular cancer 2014 — PMC4189190.
- PubMed PMID:8406456 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:14574404 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for THBS2 (Thrombospondin-2). Expected localisation Granular cytoplasmic staining in several tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A03253 at 1:100 (the product's own IHC validation image is shown). Controls (HPA): positive Adipose tissue (Adipocytes); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded rat testis tissue; fixative not specified (datasheet A03253); 2. Fixation: Image fixative and duration unreported (datasheet A03253); 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 citrate buffer, pH 6.0 , 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 anti-THBS2, 1:100 (datasheet A03253); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Secreted THBS2 may stain away from its RNA source (HPA tissue IHC); Isoform / epitope: No annotated isoforms; mature chain starts at residue 19 (UniProt); Regulation: Blood vessel–enhanced RNA expression (HPA tissue RNA). Evidence: UniProt P35442, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the plasma membrane. In addition localized to…, Datasheet A03253, PMC11178913, PMC5727893. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/THBS2/immunohistochemistry-ihc-guide.