This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
SEMA3B / Semaphorin-3B · IHC design guide
Design Immunohistochemistry for SEMA3B
Use the IHC-validated antibody at 0.5–1 µg/mL for paraffin sections (datasheet A06559-1). This guide helps interpret granular cytoplasmic and additional plasma staining using glandular and glial cells as reference patterns (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
SEMA3B 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 cytoplasm; additional plasma staining (HPA tissue IHC) | |
| Staining pattern | Granular cytoplasm in glandular and glial cells; plasma positivity (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A06559-1) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | None in HPA (detected in all 44 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 variants may uncouple tissue RNA and protein staining (HPA tissue IHC) | |
| Regulation | Enhanced RNA in adrenal gland and brain (HPA tissue RNA) | |
| Isoform / epitope | 2 isoforms; mature chain 25–749 lacks a cytoplasmic domain (UniProt) |
Section 1
Recommended SEMA3B IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: A06559-1) is followed by three published SEMA3B IHC protocols (PMC6598462; PMC8864000; PMC11371361).
| Sample | Paraffin-embedded human mammary cancer tissue; fixative not specified (datasheet A06559-1) |
| Fixation | Image fixative and duration unreported (datasheet A06559-1); 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 A06559-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A06559-1) |
| Primary antibody | Rabbit anti-SEMA3B, 0.5-1μg/ml (datasheet A06559-1) |
| Primary incubation | Overnight at 4 °C (datasheet A06559-1) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A06559-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | SEMA3B-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression with a granular pattern. Additional plasma positivity. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet: A06559-1). Published alternatives include citrate at pH 6 and a pH 9 solution (PMC6598462; PMC8864000).
Section 2
What Is the Expected SEMA3B Staining Pattern?
SEMA3B is secreted and can accumulate in the endoplasmic reticulum; it has no transmembrane segment (UniProt Q13214). In tissue sections, expect granular cytoplasmic staining, with additional plasma positivity (HPA tissue IHC). HPA reports high staining in several glandular and neural cell populations, but rates its tissue IHC evidence Uncertain because secretion complicates comparison of RNA and protein locations (HPA tissue IHC).
| Granular cytoplasmic staining in adrenal or duodenal glandular cells. | This fits the reported general pattern and High staining in those cells (HPA tissue IHC). Judge the signal against nearby tissue and the assay controls (standard IHC practice). |
| Plasma staining accompanies cellular staining. | Additional plasma positivity is reported (HPA tissue IHC). Secreted SEMA3B makes extracellular signal plausible (UniProt Q13214); plasma staining alone does not identify its producing cell. |
| Strong nuclear or sharply membrane restricted staining dominates. | That differs from HPA's granular cytoplasmic pattern and UniProt's secreted, nontransmembrane annotation (HPA tissue IHC; UniProt Q13214). Check for artefact before scoring it as SEMA3B. |
| Strong signal appears mainly in a cell type outside the reported pattern. | Possible causes include cross reactivity or endogenous detection activity (standard IHC practice). HPA lists no negative tissue, so an unlisted cell type alone cannot establish a false positive (HPA tissue IHC). |
| A reported High tissue has no specific staining. | This conflicts with the reported result but does not prove biological absence (HPA tissue IHC). Check tissue identity, positive control and detection performance (standard IHC practice). |
💡Expected SEMA3B appearanceCall a result consistent when reported High cells show granular cytoplasmic staining, possibly with plasma positivity; dominant nuclear or membrane staining warrants investigation (HPA tissue IHC; UniProt Q13214).
| Cellular versus extracellular location | SEMA3B is secreted and accumulates in the endoplasmic reticulum (UniProt Q13214). HPA reports granular cytoplasm plus plasma positivity; extracellular signal need not mark the source cell (HPA tissue IHC). |
| Choice of comparison tissue | HPA reports High staining in adrenal and duodenal glandular cells, cortical glia and cardiomyocytes, among others; ovarian stroma is Low, not negative (HPA tissue IHC). |
| Strength of the tissue evidence | HPA rates tissue IHC Uncertain and HPA030651 IHC Uncertain (HPA tissue IHC; HPA antibodies). Treat a matching pattern as supportive rather than definitive identification. |
| Processing and isoforms | The signal peptide spans residues 1–24; the annotated chain spans 25–749, with 2 isoforms (UniProt Q13214). Epitope location is unspecified, so isoform recognition cannot be inferred. |
| Glycosylation | UniProt lists glycosylation sites at 82, 124 and 427 (UniProt Q13214). Their effect on this antibody's staining or antigen retrieval is unreported. |
| IF/ICC Q: Is the same pattern established in cultured cells? | A: HPA calls SEMA3B secreted but lists no ICC-IF image cell lines or main location; HPA030651 has no ICC validation entry (HPA subcellular; HPA antibodies). An IF pattern remains unverified here. |
| Situation | Likely cause | Next action |
|---|---|---|
| Reported High tissue is blank. | Tissue mix-up or a failed staining run is possible (standard IHC practice); a blank slide alone cannot overturn HPA's reported High staining (HPA tissue IHC). | Verify the tissue and review the run's positive control, antibody application and detection steps before interpreting SEMA3B as absent (standard IHC practice). |
| Most of the section has diffuse brown color. | Nonspecific background or endogenous detection activity may obscure specific staining (standard IHC practice). | Compare a reagent omission control, review blocking and washing, then score only a distinguishable granular cellular pattern (standard IHC practice; HPA tissue IHC). |
| Nuclei are the strongest stained compartment. | This conflicts with the reported granular cytoplasmic pattern and secreted annotation (HPA tissue IHC; UniProt Q13214). | Inspect controls and counterstain, then reassess whether the nuclear color is specific before assigning a positive score (standard IHC practice). |
| A vessel or plasma rich area stains without clear cellular signal. | Additional plasma positivity is reported for secreted SEMA3B (HPA tissue IHC; UniProt Q13214). It cannot localize production to adjacent cells. | Record plasma and cellular signals separately; use the reported cell pattern when assessing cellular positivity (HPA tissue IHC). |
| Unexpected cells stain strongly. | Cross reactivity or endogenous activity is possible (standard IHC practice). HPA supplies no negative tissue designation for a decisive comparison (HPA tissue IHC). | Review morphology and detection controls, and avoid calling the cells SEMA3B positive from location alone (standard IHC practice). |
| Staining is confined to ovarian stroma at low intensity. | HPA reports ovarian stroma as Low and rates its tissue IHC evidence Uncertain (HPA tissue IHC). | Compare a reported High tissue in the same run before treating the weak signal as either a positive control or proof of assay failure (HPA tissue IHC; standard IHC practice). |
Experiment evidence
IHC & IF images showing SEMA3B staining
A06559-1 · IHCIHC analysis of SEMA3B using anti-SEMA3B antibody (A06559-1). SEMA3B was detected in paraffin-embedded section of human mammary cancer tissu
A06559-1 · IHCIHC analysis of SEMA3B using anti-SEMA3B antibody (A06559-1). SEMA3B was detected in paraffin-embedded section of human colon cancer tissue.
A06559-1 · IFIF analysis of SEMA3B using anti-SEMA3B antibody (A06559-1). SEMA3B was detected in immunocytochemical section of A431 cells. Enzyme antigen
M06559 · IFIF analysis of Semaphorin 3B/SEMA3B using anti-Semaphorin 3B/SEMA3B antibody (M06559). Semaphorin 3B/SEMA3B was detected in an immunocytoche
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000012171-SEMA3B/tissue/cerebral+cortex#imid_8104976
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000012171-SEMA3B/tissue/colon#imid_8104910
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000012171-SEMA3B/tissue/liver#imid_8104895
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000012171-SEMA3B/tissue/kidney#imid_8104904
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for SEMA3B IHC & IF
🧪Run adrenal gland first: its glandular cells should stain strongly (HPA: High in adrenal gland glandular cells). HPA detects SEMA3B in all 44 scored tissues, so there is no HPA-supported negative tissue; use no-primary and isotype controls, and assess cells without specific staining on the positive slide as internal background rather than assuming a particular cell type is SEMA3B-negative (HPA: no negative rows).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: SEMA3B is detected in all 44 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for SEMA3B; derive a cell-line control from the positive tissue's cell type (Glandular cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary (secondary-only) slide, a concentration-matched rabbit isotype control, and SEMA3B knockout tissue if available; the selected primary antibody is rabbit-derived (selected-SKU tissue-IHC caption: rabbit anti-SEMA3B). For adrenal sections, control endogenous peroxidase and biotin signal when using the caption’s SABC–DAB detection method (selected-SKU tissue-IHC caption: SABC–DAB).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU tissue-IHC caption: fixative unreported). The caption uses heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required (selected-SKU tissue-IHC caption: EDTA pH 8.0). The supplied evidence does not show that frozen sections or IF are easier, and interpretation should allow for secreted and endoplasmic reticulum localization (HPA subcellular: Secreted; UniProt Q13214: Secreted, endoplasmic reticulum).
HPA tissue IHC evidence for SEMA3B
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Caudate | Glial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | Glial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: SEMA3B is detected in all 44 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced SEMA3B IHC Tips
Use the catalog antibody’s paraffin-section example as the starting point, then assess SEMA3B staining by compartment, cell type and controls.
Which retrieval conditions should I start with for paraffin-section SEMA3B IHC?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 (datasheet A06559-1). The selected paraffin-section example used this retrieval before staining with 1 μg/mL antibody overnight at 4°C (datasheet A06559-1). If staining is weak, compare a citrate retrieval condition on matched sections while keeping antibody concentration and detection constant (standard IHC practice). Include a section processed without primary antibody to assess detection background (standard IHC practice). Evaluate granular cytoplasmic staining and any plasma signal separately, because both appear in the reported tissue profile (HPA: tissue IHC profile).
How should I assess whether fixation is affecting SEMA3B staining?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative (datasheet A06559-1). Record each specimen’s fixative and fixation duration, then compare similarly processed sections when investigating a weak or uneven result (standard IHC practice). Keep retrieval at EDTA pH 8.0 and the antibody at 1 μg/mL while assessing that variable (datasheet A06559-1). Include a concurrently stained reference section to distinguish a specimen-processing problem from a run-wide failure (standard IHC practice). Do not infer fixation tolerance from the reported tissue pattern or protein topology; neither measures fixation sensitivity (HPA: tissue IHC profile; UniProt Q13214 topology).
Which staining compartments are plausible for SEMA3B in tissue sections?
Assess granular cytoplasmic staining and plasma positivity as separate patterns; both are reported in tissue IHC (HPA: tissue IHC profile). SEMA3B is secreted and can accumulate in the endoplasmic reticulum, so intracellular and extracellular signal warrant separate annotation (UniProt Q13214 subcellular location). It has no transmembrane segment, making a sharply membrane-restricted pattern a reason to inspect specificity controls (UniProt Q13214 topology). Compare staining with nearby morphology and a section lacking primary antibody before assigning extracellular chromogen to SEMA3B (standard IHC practice). Avoid treating a stained cell as the proven source of adjacent extracellular protein, because secreted protein can separate tissue RNA and protein locations (HPA: reliability description).
Can this antibody distinguish SEMA3B isoforms or processing states by IHC?
Do not assign an IHC pattern to a specific isoform without an epitope map and isoform-specific validation; 2 isoforms are listed (UniProt Q13214 isoforms; standard IHC practice). The annotated signal peptide spans residues 1–24, followed by a 25–749 chain (UniProt Q13214 processing). The protein also has annotated glycosylation sites at residues 82, 124 and 427, which may matter when choosing independent epitopes for comparison (UniProt Q13214 glycosylation; standard IHC practice). Request the antibody’s immunogen or epitope information before interpreting discordant staining as isoform selectivity (standard IHC practice). Compare independently validated epitopes on matched sections if the distinction is essential (standard IHC practice).
How can I investigate an IHC pattern with multiplex IF?
Treat IF as a separate assay requiring its own validation; the supplied HPA record lists no ICC/IF image-bearing cell lines (HPA: subcellular record). For tissue multiplexing, pair SEMA3B with a marker identifying the glandular or glial cell population being assessed, rather than relying on overlap alone (HPA: tissue IHC positives; standard IF practice). Choose spectrally separated fluorophores and consider a far-red channel after checking tissue autofluorescence (standard IF practice). If testing an intracellular epitope, include controlled permeabilisation; for accessible extracellular signal, compare an unpermeabilised condition (UniProt Q13214: secreted, no transmembrane segment; standard IF practice). Interpret IF alongside matched chromogenic IHC morphology and negative controls (standard IHC/IF practice).
How do I separate genuine SEMA3B signal from chromogenic background?
Run a section without primary antibody and inspect it for staining introduced by the detection system (standard IHC practice). The selected example used a biotinylated secondary antibody, a streptavidin–biotin complex and DAB, so assess background with that complete detection sequence (datasheet A06559-1). Include an endogenous peroxidase block and evaluate tissue-dependent biotin background when using this workflow (standard IHC practice). Inspect edges, folds and damaged areas before scoring diffuse chromogen as extracellular SEMA3B (standard IHC practice; UniProt Q13214: secreted). If background rises with antibody concentration, compare matched sections under the documented 1 μg/mL condition while keeping detection constant (datasheet A06559-1; standard IHC practice).
What should I score when SEMA3B appears both intracellular and extracellular? ⚠ ANSWER MARKED FOR VERIFICATION
Predefine separate scores for cellular granular cytoplasmic staining and plasma or other extracellular signal, since the reported profile includes both (HPA: tissue IHC profile). For cells, record the percentage positive and an intensity-based H-score from 0–300 within a defined cell population (standard IHC practice). For extracellular staining, measure positive area or optical density per mm² of eligible tissue using consistent image settings (standard IHC practice). Normalise cellular results to the number of evaluable cells and area measurements to evaluable tissue area (standard IHC practice). Exclude folds, necrosis and poorly preserved regions using rules set before reviewing groups (standard IHC practice).
When should I question a positive SEMA3B IHC result?
Give greater weight to a reproducible granular cytoplasmic pattern in a plausible cell population, while recording plasma signal separately (HPA: tissue IHC profile; standard IHC practice). Glandular cells in several tissues and glial cells in the caudate and cerebral cortex are reported as high staining populations, but the overall tissue-IHC reliability is uncertain (HPA: positive tissues and reliability). Question a purely sharp membrane outline because SEMA3B lacks a transmembrane segment (UniProt Q13214 topology). Check edge staining, necrosis and signal in the no-primary control before calling weak diffuse DAB positive (standard IHC practice). A positive extracellular area does not establish which nearby cell produced this secreted protein (UniProt Q13214 subcellular location; HPA: reliability description).
Boster reagents
Best SEMA3B / Semaphorin-3B IHC Antibodies
SEMA3B antibodies have IHC data from human cancer and mouse brain paraffin sections (A06559-1 image captions), plus IF/ICC data from human A431 and HeLa cells (A06559-1 and M06559 image captions).
Real IHC data
Anti-Semaphorin 3B/SEMA3B Antibody ®
Cat # A06559-1
Real IF data
Anti-Semaphorin 3B/SEMA3B Antibody ® (monoclonal, 9C4F7)
Cat # M06559
A06559-1 is listed for IHC and IF/ICC in human, mouse and rat (catalog applications and reactivity); its images show paraffin-section IHC in human mammary and colon cancer and mouse brain, plus IF/ICC in A431 cells (A06559-1 image captions). M06559 is listed for IF/ICC, but not IHC, with human, mouse and rat reactivity (catalog applications and reactivity); its IF image shows HeLa cells (M06559 image caption).
Which to pick: Choose rabbit A06559-1 for tissue IHC because its own images document human and mouse paraffin sections; the fixative is unreported (A06559-1 image captions). For IF/ICC, both A06559-1 and mouse monoclonal M06559 have cell images, in A431 and HeLa respectively (catalog host and clone; A06559-1 and M06559 image captions). For cross-species tissue IHC, A06559-1 has human and mouse images and listed rat reactivity, though no rat IHC image is supplied (A06559-1 image captions; catalog reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q13214 (SEM3B_HUMAN, Semaphorin-3B).
- Human Protein Atlas. SEMA3B tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. SEMA3B subcellular location (ICC-IF): Secreted.
- Human Protein Atlas. SEMA3B antibody validation summary (1 antibodies).
- Expression of Semaphorin 3B (SEMA3B) in Various Grades of Endometrial Cancer. Medical science monitor : international medical journal of experimental and clinical research 2019 — PMC6598462.
- Prevalence of neural epidermal growth factor-like 1- and exostosin 1/exostosin 2-associated membranous nephropathy: a single-center retrospective study in Japan. Scientific reports 2022 — PMC8864000.
- SEMA3B inhibits TGFβ-induced extracellular matrix protein production and its reduced levels are associated with a decline in lung function in IPF. American journal of physiology. Cell physiology 2024 — PMC11371361.
- Semaphorin 3B-Membranous Nephropathy in Adults. Kidney international reports 2026 — PMC13223841.
- PubMed PMID:8633026 — UniProt-cited evidence.
- PubMed PMID:16641997 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for SEMA3B (Semaphorin-3B). Expected localisation Granular cytoplasm; additional plasma staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A06559-1). Validated IHC antibody: A06559-1 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative None in HPA (detected in all 44 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human mammary cancer tissue; fixative not specified (datasheet A06559-1); 2. Fixation: Image fixative and duration unreported (datasheet A06559-1); 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 A06559-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A06559-1); 8. Primary antibody: Rabbit anti-SEMA3B, 0.5-1μg/ml (datasheet A06559-1); 9. Primary incubation: Overnight at 4 °C (datasheet A06559-1). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Secreted variants may uncouple tissue RNA and protein staining (HPA tissue IHC); Isoform / epitope: 2 isoforms; mature chain 25–749 lacks a cytoplasmic domain (UniProt); Regulation: Enhanced RNA in adrenal gland and brain (HPA tissue RNA). Evidence: UniProt Q13214, HPA tissue IHC · Uncertain, HPA ICC-IF · Secreted, Datasheet A06559-1, PMC6598462, PMC8864000. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/SEMA3B/immunohistochemistry-ihc-guide.