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THBS4 / Thrombospondin-4 · IHC design guide
Design Immunohistochemistry for THBS4
Plan THBS4 paraffin IHC around extracellular deposits and cytoplasmic staining (HPA tissue IHC). Lung macrophages and breast glandular cells show medium staining (HPA tissue IHC); titrate the IHC-validated antibody within 1:50–1:200 (datasheet: A05024).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
THBS4 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 | Extracellular deposits and cytoplasm (HPA tissue IHC) | |
| Staining pattern | Extracellular deposits and cytoplasmic signal in several tissues (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Breast+4 more · see all | |
| Negative control | Adrenal gland+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Secreted THBS4 may stain away from its site of RNA expression (HPA tissue IHC) | |
| Regulation | Staining regulation is not established (UniProt) | |
| Isoform / epitope | No annotated isoforms; signal peptide removed; no cytoplasmic tail (UniProt) |
Section 1
Recommended THBS4 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by four published THBS4 chromogenic IHC methods, covering paraffin sections and fixed coverslip cells (PMC2946314; PMC5450725; PMC12764563; PMC6839882).
| Sample | Paraffin-embedded human lung tissue; fixative not specified (datasheet A05024) |
| Fixation | Image fixative and duration unreported (datasheet A05024); 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-THBS4, 1:50 recommended; image 1:200 (datasheet A05024) |
| 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 | THBS4-positive staining in glandular cells of breast (HPA tissue IHC: Medium). HPA tissue profile: Extracellular deposits and cytoplasmic expression in several tissues. No signal in the no-primary control. |
💡Decision noteFor paraffin sections, start with citrate pH 6.0 HIER at 95–98 °C for 20 min (page antigen retrieval).
Section 2
What Is the Expected THBS4 Staining Pattern?
In paraffin-section IHC, expect THBS4 in extracellular deposits and the cytoplasm of selected cells, including breast and fallopian-tube glandular cells, lung macrophages, nasopharyngeal respiratory epithelium, and testicular Leydig cells (HPA tissue IHC: Medium). Its secreted, extracellular-matrix and endoplasmic-reticulum locations fit that pattern; it has no transmembrane segment (UniProt P35443). HPA rates the tissue staining Approved but reports low consistency with RNA expression (HPA tissue IHC).
| Extracellular deposits with cytoplasmic staining in the listed cell types. | This matches the reported tissue profile; each listed cell type has Medium staining (HPA tissue IHC). Deposits are compatible with a secreted matrix protein, while cytoplasm is compatible with its intracellular locations (UniProt P35443). |
| Dominant nuclear staining or a crisp, continuous plasma-membrane outline. | Treat this as suspect and compare with a control section. HPA reports extracellular deposits and cytoplasm, and UniProt reports no transmembrane segment or nuclear location (HPA tissue IHC; UniProt P35443 topology and subcellular location). |
| Strong staining in a cell population reported as not detected. | Check antibody specificity and endogenous chromogen-producing activity before assigning THBS4. For example, HPA reports adrenal glandular cells and bone-marrow hematopoietic cells as Not detected; an unexpected signal alone does not identify its cause (HPA tissue IHC; general IHC practice). |
| Diffuse color across cells, matrix, and section edges without anatomical structure. | This does not resemble HPA's extracellular-deposit and cytoplasmic profile (HPA tissue IHC). Review background in the negative control, blocking, washes, and detection chemistry; these are general IHC checks, not documented THBS4-specific effects (general IHC practice). |
| No signal in a section containing an HPA-listed Medium-staining cell type. | Check that the relevant cells are present, then review the antibody, retrieval, and detection controls (HPA tissue IHC; general IHC practice). HPA's Approved tissue profile has low RNA–protein consistency, so a single absent result needs cautious interpretation (HPA tissue IHC). |
💡Expected THBS4 appearanceCall a result consistent with THBS4 when extracellular deposits and cytoplasm stain in an HPA-listed Medium cell population; dominant nuclear or continuous membrane staining is suspect (HPA tissue IHC; UniProt P35443 topology).
| Compartment and processing | THBS4 has a signal peptide at residues 1–26 and a mature chain at 27–961; UniProt places it in the ER and extracellular matrix (UniProt P35443). Interpret deposits and cellular staining together, without assuming that every cell containing THBS4 RNA retains its secreted protein (HPA tissue IHC). |
| Tissue choice and signal strength | Breast and fallopian-tube glandular cells, lung macrophages, nasopharyngeal respiratory epithelium, and Leydig cells are Medium; heart cardiomyocytes and soft-tissue fibroblasts are Low (HPA tissue IHC). The listed Low populations offer a less conspicuous visual check than the listed Medium populations (HPA tissue IHC). |
| Antibody evidence | CAB004597 has Approved IHC status; HPA042426 has Approved ICC status, with no IHC status supplied for that antibody (HPA antibodies). The tissue profile is Approved but has low consistency between antibody staining and RNA data; secretion complicates location matching (HPA tissue IHC). |
| IF/ICC: what pattern is reported? | Vesicles are the approved ICC-IF location, with images listed for AF22, Rh30, and U2OS (HPA subcellular). This answers the IF/ICC interpretation question; the paraffin IHC pattern remains extracellular deposits and cytoplasm in tissue (HPA tissue IHC). |
| Retrieval and detection | When troubleshooting an IHC run, review the chosen retrieval and chromogenic detection controls (general IHC practice). Neither HPA tissue IHC nor UniProt P35443 supplies a THBS4-specific fixation effect or retrieval condition. |
| Situation | Likely cause | Next action |
|---|---|---|
| No staining in an HPA-listed Medium population. | The relevant cells may be absent from the section, or the IHC workflow may have failed (HPA tissue IHC; general IHC practice). | Confirm the cell population, then check the IHC-validated antibody and run controls; review retrieval and detection steps without assuming THBS4-specific fixation sensitivity (HPA antibodies; general IHC practice). |
| Only faint staining in heart cardiomyocytes or soft-tissue fibroblasts. | Both populations are reported as Low, so faint staining can fit the reference pattern (HPA tissue IHC). | Compare with an HPA-listed Medium population processed in the same run before treating faint signal as an assay failure (HPA tissue IHC; general IHC practice). |
| Diffuse chromogen obscures extracellular deposits and cells. | Excess background can hide the tissue pattern; the image alone cannot establish whether primary binding or detection chemistry caused it (HPA tissue IHC; general IHC practice). | Compare negative controls, inspect washes and blocking, and reassess whether deposits and cytoplasm remain distinguishable (general IHC practice; HPA tissue IHC). |
| Strong nuclear or continuous membrane staining dominates. | Those compartments do not match HPA's tissue profile or UniProt's listed locations and topology (HPA tissue IHC; UniProt P35443). | Compare an HPA-listed Medium population and a negative control; investigate antibody specificity and detection background before scoring the signal as THBS4 (HPA tissue IHC; general IHC practice). |
| A cell population listed as Not detected stains strongly. | Cross-reactivity or endogenous detection activity is possible, but staining alone cannot distinguish them (HPA tissue IHC; general IHC practice). | Check a no-primary control and the detection system, then compare the same run with an HPA-listed Medium population (general IHC practice; HPA tissue IHC). |
| Extracellular deposits appear away from the cell type expected to produce THBS4. | A secreted protein can occupy a different tissue location from its RNA source; HPA specifically flags this correlation problem (UniProt P35443; HPA tissue IHC). | Score deposit location and cellular staining separately, and use the reported IHC pattern rather than RNA location alone to interpret the section (HPA tissue IHC). |
Experiment evidence
IHC & IF images showing THBS4 staining
A05024 · IHCImmunohistochemical analysis of paraffin-embedded human-lung, antibody was diluted at 1:200
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000113296-THBS4/tissue/cerebral+cortex#imid_4966164
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000113296-THBS4/tissue/colon#imid_4966106
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000113296-THBS4/tissue/liver#imid_4966107
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000113296-THBS4/tissue/kidney#imid_4966100
HPA · ICC-IF AF22Human Protein Atlas immunofluorescence, AF22, antibody HPA042426; green = THBS4, blue = nucleus, red = microtubules.
HPA · ICC-IF AF22Human Protein Atlas immunofluorescence, AF22, antibody HPA042426; green = THBS4, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for THBS4 IHC & IF
🧪Run lung first: macrophages should show THBS4 staining at the Medium level (HPA: lung macrophages); the catalog antibody’s paraffin-section image also uses human lung (caption: A05024, 1:200). Run adrenal gland as a negative tissue (HPA: adrenal gland glandular cells, Not detected), and use neighboring cells without specific staining on the lung slide as internal negative comparators rather than assuming every nonmacrophage is negative.
Positive control tissue: Breast (Glandular cells, HPA Medium)
Negative control tissue: Adrenal gland (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show THBS4 in AF22, Rh30, U2OS, with annotated localisation: Vesicles (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; use a host- and immunoglobulin-class-matched isotype control for a monoclonal primary or normal host IgG for a polyclonal primary; and confirm specificity with knockout tissue or peptide blocking where available (standard IHC practice). Quench endogenous peroxidase for chromogenic staining and check lung macrophages for pigment that could be mistaken for signal (standard IHC practice; HPA: lung macrophages).
⚠️Feasibility: A target-specific fixation window and retrieval dependency are unreported in the supplied evidence; the selected 1:200 paraffin-section caption does not state a fixative (caption: A05024). The evidence does not establish that frozen sections or IF are easier than paraffin IHC; ICC-IF images show vesicular localization in AF22, Rh30 and U2OS cells (HPA: subcellular). In lung, distinguish macrophage pigment from chromogenic signal when scoring (HPA: lung macrophages; standard IHC practice).
HPA tissue IHC evidence for THBS4
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low consistency between antibody staining and RNA expression data. At least one protein variant secreted, tissue location of RNA and protein might differ and correlation is complex.). 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 |
|---|---|---|---|---|
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | Medium | Protein (IHC) | HPA → |
| Lung | Macrophages | Medium | Protein (IHC) | HPA → |
| Nasopharynx | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Testis | Leydig cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced THBS4 IHC Tips
Troubleshoot THBS4 staining by assessing retrieval, compartment, and cell identity alongside matched controls (UniProt P35443; HPA tissue IHC).
What should I change if THBS4 staining is weak after retrieval?
Start with citrate pH 6.0 heat-induced antigen retrieval at 95–98 °C for 20 min on paraffin sections (page retrieval specification). Compare a retrieved section with an otherwise matched section processed without retrieval, keeping antibody dilution and chromogen development constant (standard IHC practice). The selected antibody has a paraffin-embedded human lung image at 1:200, but its caption does not report retrieval conditions (A05024 caption). If staining remains weak, test a more alkaline retrieval buffer as a fallback on adjacent sections, while checking whether increased signal also raises extracellular or tissue background (standard IHC practice; UniProt P35443 localisation).
How can I troubleshoot fixation-related variation in THBS4 IHC?
THBS4-specific sensitivity to fixation duration or fixative chemistry is unknown because no target-specific fixation evidence is supplied (provided fixation evidence). The selected antibody caption identifies paraffin-embedded human lung and a 1:200 dilution, but does not state the fixative (A05024 caption). Record each specimen’s fixative, fixation interval, processing history, and section age, then compare staining in specimens prepared under matched conditions (standard IHC practice). When comparing runs, hold citrate pH 6.0 retrieval at 95–98 °C for 20 min constant so a processing difference is easier to identify (page retrieval specification; standard IHC practice).
Which THBS4 staining compartments should I expect in paraffin sections?
Assess extracellular deposits and cytoplasmic signal separately, because both occur in the tissue IHC profile (HPA tissue IHC). THBS4 is annotated as secreted into extracellular space and matrix, and also localised to endoplasmic and sarcoplasmic reticulum (UniProt P35443 localisation). An exclusively sharp cell-surface rim would need scrutiny because THBS4 has no transmembrane segment (UniProt P35443 topology). In lung, compare candidate positive cells with macrophage morphology because macrophages show medium staining in the tissue profile (HPA: Lung macrophages, Medium). Check adjacent sections and a negative control before attributing diffuse interstitial chromogen to deposited THBS4 (standard IHC practice; HPA tissue IHC).
Could epitope placement explain different intracellular and matrix staining?
First obtain the antibody’s immunogen or epitope coordinates; the supplied caption gives a 1:200 dilution but no epitope position (A05024 caption). THBS4 has a signal peptide at residues 1–26 and a mature chain at 27–961, so an epitope confined to the signal peptide would require special caution when interpreting secreted protein (UniProt P35443 processing). Annotated glycosylation sites at 612 and 941 and the C-terminal domain at 731–945 provide landmarks for reviewing epitope location (UniProt P35443). No isoforms are listed in the supplied record; use a second antibody with a documented, distinct epitope to investigate discordant patterns (UniProt P35443 isoforms; standard IHC validation practice).
How should I check an ambiguous THBS4 IHC pattern with multiplex IF?
Use IF as a follow-up comparison, pairing THBS4 with a marker that identifies the expected cells; lung macrophages are one documented positive population (HPA: Lung macrophages, Medium). Choose fluorophores after examining unstained tissue in each channel, and favour a channel with low tissue autofluorescence for the weaker signal (standard IF practice). For an intracellular epitope, assess mild permeabilisation; for an extracellular epitope, compare staining without permeabilisation, using the antibody’s documented epitope location to guide that choice (standard IF practice; UniProt P35443 localisation). Interpret puncta alongside the reported vesicular IF localisation, while keeping extracellular deposits distinct from cellular signal (HPA subcellular; HPA tissue IHC).
What causes widespread brown staining around THBS4-positive structures?
Begin by comparing a no-primary control, a matched tissue section, and the THBS4 stain developed for the same duration (standard chromogenic IHC practice). Block endogenous peroxidase before horseradish peroxidase detection, and shorten DAB development if the no-primary control also turns brown (standard chromogenic IHC practice). Extracellular THBS4 deposits are plausible, so diffuse signal alone is insufficient grounds to call background (HPA tissue IHC; UniProt P35443 localisation). Check whether staining follows tissue folds, section edges, or necrotic areas, then compare intact regions at the same exposure and scoring threshold (standard IHC practice). Reassess antibody concentration using the caption’s 1:200 reference point (A05024 caption).
How should I quantify THBS4 when cells and matrix both stain? ⚠ ANSWER MARKED FOR VERIFICATION
Define cellular and extracellular regions before scoring, since the tissue profile includes cytoplasmic expression and extracellular deposits (HPA tissue IHC). For identified cells, record percent positive and intensity, or calculate an H-score from prespecified intensity categories; normalise to the number of eligible cells assessed (standard IHC quantification practice). For matrix deposits, measure stained area or deposit density per mm² of eligible tissue, excluding folds and necrosis using the same rule across sections (standard IHC quantification practice). Report the compartments separately and apply one chromogen threshold, magnification, and region-selection rule across the comparison set (standard IHC quantification practice; HPA tissue IHC).
When is an apparent THBS4-positive focus likely to be artefact?
A credible focus should fit a documented compartment and, where cell identity is claimed, a documented cell population (UniProt P35443 localisation; HPA tissue IHC). In lung, medium macrophage staining is reported; a claim that another lung cell type is positive needs independent cell identification (HPA: Lung macrophages, Medium). Treat a sharp membrane-only pattern cautiously because THBS4 lacks a transmembrane segment, while extracellular deposits and cytoplasmic signal are documented (UniProt P35443 topology; HPA tissue IHC). Compare edge and necrotic staining with intact tissue, and check a no-primary section for endogenous enzyme or detection-related colour before calling a focus positive (standard chromogenic IHC practice).
Boster reagents
Best THBS4 / Thrombospondin-4 IHC Antibodies
A05024 is listed for IHC and IF in human, mouse, and rat, with a real IHC image of paraffin-embedded human lung (catalog applications/reactivity; IHC image caption).
Real IHC data
Anti-Thrombospondin 4 THBS4 Antibody
Cat # A05024
A05024 has an IHC image of paraffin-embedded human lung at 1:200 (catalog IHC image caption). A05024 is also listed for IF and for human, mouse, and rat reactivity; no IF image is supplied (catalog applications/reactivity; IF image alts).
Which to pick: Choose A05024 for paraffin-section tissue IHC: its own image shows human lung at 1:200, and the fixative is unreported (catalog IHC image caption). For IF/ICC planning, A05024 is a rabbit polyclonal listed for IF; ICC is not separately listed, and no IF image is supplied (catalog host/clonality; applications; IF image alts). For cross-species work, A05024 lists human, mouse, and rat reactivity, while its supplied IHC image documents human lung only (catalog reactivity; IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P35443 (TSP4_HUMAN, Thrombospondin-4).
- Human Protein Atlas. THBS4 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. THBS4 subcellular location (ICC-IF): Localized to vesicles..
- Human Protein Atlas. THBS4 antibody validation summary (2 antibodies).
- Thrombospondin-4 is a putative tumour-suppressor gene in colorectal cancer that exhibits age-related methylation. BMC cancer 2010 — PMC2946314.
- Effects of hypoxia on differentiation of menstrual blood stromal stem cells towards tenogenic cells in a co-culture system with Achilles tendon cells. Experimental and therapeutic medicine 2017 — PMC5450725.
- Thrombospondin-4 regulates apoptosis of vascular smooth muscle cells after artery transplanted into vein. Scientific reports 2025 — PMC12764563.
- The clinicopathological significance of Thrombospondin-4 expression in the tumor microenvironment of gastric cancer. PloS one 2019 — PMC6839882.
- PubMed PMID:8350346 — UniProt-cited evidence.
- PubMed PMID:7852353 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for THBS4 (Thrombospondin-4). Expected localisation Extracellular deposits and cytoplasm (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A05024 at 1:50 recommended; image 1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Breast (Glandular cells); negative Adrenal gland. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung tissue; fixative not specified (datasheet A05024); 2. Fixation: Image fixative and duration unreported (datasheet A05024); 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-THBS4, 1:50 recommended; image 1:200 (datasheet A05024); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Secreted THBS4 may stain away from its site of RNA expression (HPA tissue IHC); Isoform / epitope: No annotated isoforms; signal peptide removed; no cytoplasmic tail (UniProt); Regulation: Staining regulation is not established (UniProt). Evidence: UniProt P35443, HPA tissue IHC · Approved, HPA ICC-IF · vesicles., Datasheet A05024, PMC2946314, PMC5450725. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/THBS4/immunohistochemistry-ihc-guide.