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SEMA4F / Semaphorin-4F · IHC design guide
Design Immunohistochemistry for SEMA4F
SEMA4F staining is reported in the retinal nerve fiber layer (HPA tissue IHC), while membrane localization is annotated at the protein level (UniProt). Use this guide to plan paraffin-section IHC with antibody A11873-2 and interpret staining in light of reported off-target binding (datasheet A11873-2; HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
SEMA4F 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 | Retinal nerve fibers (HPA tissue IHC); membrane expected (UniProt) | |
| Staining pattern | Medium staining in retinal nerve fiber layer; finer pattern unreported (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A11873-2) | |
| Positive control | Retina | |
| Negative control | Caudate+4 more · see all |
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 A11873-2) | |
| Caveat | Presumed off-target staining was disregarded (HPA tissue IHC) | |
| Regulation | Regulation not annotated (UniProt) | |
| Isoform / epitope | 2 isoforms; check whether the epitope is extracellular or cytoplasmic (UniProt) |
Section 1
Recommended SEMA4F IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by one published SEMA4F IHC protocol using ovarian tumour and adjacent non-tumour tissues (PMC10525231).
| Sample | Paraffin-embedded human colon adenocarcinoma tissue; fixative not specified (datasheet A11873-2) |
| Fixation | Image fixative and duration unreported (datasheet A11873-2); 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 A11873-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A11873-2) |
| Primary antibody | Rabbit anti-SEMA4F, 2-5 μg/ml (datasheet A11873-2) |
| Primary incubation | Overnight at 4 °C (datasheet A11873-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A11873-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | SEMA4F-positive staining in nerve fiber layer of retina (HPA tissue IHC: Medium). HPA tissue profile: Expression in nerve fibers in retina. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (datasheet A11873-2). The published article does not specify retrieval (PMC10525231).
Section 2
What Is the Expected SEMA4F Staining Pattern?
SEMA4F is a membrane protein with an extracellular region and a cytoplasmic tail; UniProt also places it at the postsynaptic density, perikaryon and dendrites (UniProt O95754 localization and topology). In tissue IHC, the clearest reported signal is medium staining of retinal nerve fibers (HPA: retina, nerve fiber layer). Treat this as a pattern to compare with controls, since HPA rates its IHC evidence Approved and notes presumed off target binding (HPA: IHC reliability).
| Medium staining follows nerve fibers in the retinal nerve fiber layer, with little nuclear signal (HPA: retina, nerve fiber layer; UniProt O95754 topology). | This matches the reported retinal IHC pattern and is compatible with a membrane-associated protein (HPA: retina, nerve fiber layer; UniProt O95754 localization). Assess staining alongside section morphology and controls; an Approved rating supports use of the observation but does not make every stained structure target-specific (HPA: IHC reliability). |
| Predominantly nuclear staining appears, or a broad intracellular signal obscures cell borders and fibers. | That pattern does not match SEMA4F's annotated membrane, dendritic and synaptic locations (UniProt O95754 localization). Check a control section and the detection background before calling it positive; the location mismatch alone cannot identify the precise artefact (general IHC practice). |
| Strong staining appears in cells HPA reports as undetected, such as caudate glia or colonic glandular cells (HPA: caudate and colon). | Interpret cautiously: those cell-specific HPA observations do not predict a strong signal (HPA: caudate and colon). Consider cross-reactivity or endogenous detection activity, and compare no-primary and tissue controls before assigning SEMA4F positivity (general IHC practice). |
| A uniform haze or widespread chromogen deposit covers tissue structures and makes nerve fibers hard to distinguish. | This is background rather than the localized retinal pattern reported by HPA (HPA: retina, nerve fiber layer). Review blocking, washes, detection reagents and counterstain with a no-primary control; these are general IHC checks, not documented SEMA4F-specific effects (general IHC practice). |
| No discernible nerve fiber signal appears in a retinal section expected to show medium staining (HPA: retina, nerve fiber layer). | The result is inconclusive until tissue quality, antibody application and chromogenic detection have been checked (general IHC practice). HPA's observation provides a comparator, not a guarantee that every retinal specimen or run will be positive (HPA: retina, nerve fiber layer). |
💡Expected SEMA4F appearanceA convincing positive is localized, medium retinal nerve fiber layer staining consistent with membrane-associated SEMA4F; dominant nuclear staining, uniform haze or strong signal in HPA-undetected cells calls for control-based review (HPA: retina and listed negative tissues; UniProt O95754 localization; general IHC practice).
| Tissue and cell context | Retinal nerve fibers provide the clearest supplied IHC reference at medium intensity; HPA reports low staining in hippocampal glia and several other cell groups, and no detected staining in its listed negative groups (HPA: tissue IHC). A low or negative HPA entry is a comparator for that sampled cell group, not proof that an entire organ cannot express SEMA4F. |
| Topology and epitope location | SEMA4F spans residues 660–680, with residues 35–659 extracellular and 681–770 cytoplasmic (UniProt O95754 topology). The supplied record does not locate the IHC antibody's epitope, so topology alone cannot predict which preparation or retrieval condition will reveal it. |
| Protein forms | UniProt lists 2 isoforms, Long and Short, plus a signal peptide at residues 1–34 (UniProt O95754 isoforms and processing). Without the antibody epitope and isoform coverage, the staining record cannot distinguish isoforms or attribute differences between sections to processing. |
| Strength of IHC validation | HPA marks antibody HPA064095 as IHC Approved, while reporting presumed off target binding that was disregarded (HPA: antibody validation and IHC reliability). Use the reported retinal pattern as a qualified reference; this rating does not establish specificity for every tissue or unexpected compartment. |
| IF/ICC evidence boundary | HPA summarizes the subcellular location as membrane but supplies no ICC-IF image-bearing cell lines and no ICC validation entry for the listed antibody (HPA: subcellular summary and antibody validation). This supports a location comparison, not a demonstrated IF/ICC staining pattern or protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| Retinal nerve fibers show no chromogenic signal (HPA: retina, nerve fiber layer). | A failed run or an unsuitable section is possible; the supplied sources do not identify a SEMA4F-specific fixation or retrieval failure (general IHC practice; HPA: tissue IHC). | Check section integrity and the run's positive and detection controls. Review the catalog antibody's stated IHC-P conditions before changing retrieval or dilution (general IHC practice). |
| Staining is mainly nuclear. | Nuclear predominance conflicts with annotated membrane and neuronal process locations, but does not by itself identify the cause (UniProt O95754 localization). | Compare the same tissue with a no-primary control, inspect morphology and review detection background before scoring nuclei as positive (general IHC practice). |
| Strong signal appears in an HPA-undetected cell group (HPA: listed negative tissues). | Cross-reactivity or endogenous detection activity could account for unexpected staining; HPA also notes presumed off target binding in its IHC assessment (general IHC practice; HPA: IHC reliability). | Check a no-primary control and compare the signal with the retinal reference pattern. Report the unexpected cell type separately rather than treating intensity alone as confirmation (general IHC practice; HPA: retina). |
| Diffuse brown deposit masks tissue architecture. | Background from blocking, washes or detection can obscure localized staining; no target-specific mechanism is established here (general IHC practice). | Review reagent-only or no-primary controls, blocking and wash steps, then reassess whether the retinal nerve fiber layer remains distinguishable (general IHC practice; HPA: retina). |
| A faint signal appears in a tissue HPA labels low (HPA: low-staining cell groups). | Low HPA staining is compatible with a weak observed signal, but a faint deposit can also reflect background (HPA: tissue IHC; general IHC practice). | Score the specified cell group and compartment against matched background controls; avoid converting a weak, unlocalized deposit into a positive call (general IHC practice). |
| Can IF/ICC confirm the IHC pattern? | HPA's membrane summary has no accompanying ICC-IF image-bearing cell lines, and its listed antibody has no ICC validation entry (HPA: subcellular summary and antibody validation). | Use the separate IF/ICC guide for assay design. Here, describe membrane localization as a UniProt-supported expectation and keep IF/ICC claims distinct from the observed retinal IHC result (UniProt O95754 localization; HPA: retina). |
Experiment evidence
IHC & IF images showing SEMA4F staining
A11873-2 · IHCIHC analysis of SEMA4F using anti-SEMA4F antibody (A11873-2). SEMA4F was detected in a paraffin-embedded section of human colon adenocarcino
A11873-2 · IHCIHC analysis of SEMA4F using anti-SEMA4F antibody (A11873-2). SEMA4F was detected in a paraffin-embedded section of human larynx squamous ce
A11873-2 · IFIF analysis of SEMA4F using anti-SEMA4F antibody (A11873-2). SEMA4F was detected in an immunocytochemical section of U2OS cells. Enzyme anti
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000135622-SEMA4F/tissue/cerebral+cortex#imid_19405203
HPA · RetinaHuman Protein Atlas IHC, Retina. Source: https://www.proteinatlas.org/ENSG00000135622-SEMA4F/tissue/retina#imid_863
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000135622-SEMA4F/tissue/colon#imid_19405129
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000135622-SEMA4F/tissue/liver#imid_19405122
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for SEMA4F IHC & IF
🧪Run retina first: the nerve fiber layer should stain at a medium level (HPA: retina, nerve fiber layer, Medium). Use colon glandular cells as a negative tissue control (HPA: colon, glandular cells, Not detected); on the retina slide, treat cells outside the annotated nerve fiber layer as candidate internal negatives and confirm that they remain at background rather than assuming their status from the HPA row.
Positive control tissue: Retina (Nerve fiber layer, HPA Medium)
Negative control tissue: Caudate (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for SEMA4F; derive a cell-line control from the positive tissue's cell type (Nerve fiber layer) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control; rabbit IgG matched to the primary antibody’s clonality and concentration as an isotype control (selected-SKU caption: rabbit primary); and SEMA4F knockout tissue or a validated peptide-block control. Quench endogenous peroxidase for chromogenic detection and use the no-primary slide to distinguish retinal pigment from DAB staining (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU paraffin-section caption does not state a fixative (selected-SKU caption: fixative not stated). The caption uses heat retrieval in pH 8.0 EDTA, but does not establish that retrieval is required (selected-SKU caption: EDTA retrieval). No matched evidence shows that frozen sections or IF are easier; assess retinal pigment when scoring chromogenic staining (standard IHC practice).
HPA tissue IHC evidence for SEMA4F
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Presumed off target binding observed and disregarded. External characterization data supports antibody staining.). 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 |
|---|---|---|---|---|
| Retina | Nerve fiber layer | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Colon | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced SEMA4F IHC Tips
Troubleshoot SEMA4F staining in paraffin sections with chromogenic IHC, using the catalog antibody’s tissue image and independent localisation evidence.
Which retrieval condition should I try first for weak SEMA4F staining?
Start with heat-mediated retrieval in EDTA at pH 8.0 (datasheet A11873-2). That condition preceded SEMA4F staining in a paraffin section of human colon adenocarcinoma with the catalog antibody (datasheet A11873-2); the caption does not report the fixative (datasheet A11873-2). Keep section thickness, heating, cooling, and detection conditions matched across a small retrieval comparison, then judge signal against a no-primary control (standard IHC practice). If staining remains weak, test another retrieval buffer as a fallback on adjacent sections, while checking whether tissue damage or diffuse background increases (standard IHC practice).
Could fixation explain weak or uneven SEMA4F staining?
The catalog image documents a paraffin section but gives no fixative, so target-specific fixation sensitivity is unknown (datasheet A11873-2). Record fixation and processing history for each specimen, and compare sections with matched handling before attributing a difference to SEMA4F expression (standard IHC practice). When fixation history varies, assess morphology and internal staining controls alongside the signal, since tissue preservation and epitope access can affect IHC generally (standard IHC practice). Keep retrieval, primary concentration, and development time constant during that comparison; the demonstrated primary condition was 2 μg/ml overnight at 4°C (datasheet A11873-2).
Where should convincing SEMA4F staining appear in a section?
Prioritise cell-surface and process-associated staining: SEMA4F is annotated at the cell membrane, postsynaptic density, perikaryon, and dendrites (UniProt O95754 subcellular location). Its extracellular residues 35–659 and cytoplasmic residues 681–770 flank a transmembrane segment at 660–680 (UniProt O95754 topology). In retina, the reported tissue-IHC signal is medium in the nerve fiber layer (HPA tissue IHC), which supports examining fiber-rich structures when that tissue is available. Interpret diffuse nuclear or uniformly luminal staining cautiously, and compare it with morphology and no-primary controls before assigning it to SEMA4F (standard IHC practice; UniProt O95754 subcellular location).
How could isoforms or epitope position change my staining pattern?
SEMA4F has Long and Short isoforms, but the supplied record does not map this antibody’s epitope to either one (UniProt O95754 isoforms; datasheet A11873-2). Ask where the immunogen lies before treating a negative section as absence of both isoforms (standard IHC practice). The precursor has a signal sequence at 1–34, an extracellular region at 35–659, and a cytoplasmic tail at 681–770 (UniProt O95754 processing and topology). Extracellular glycosylation sites at 64, 133, and 509 make epitope location relevant when comparing retrieval conditions, although their effect on this antibody has not been established (UniProt O95754 glycosylation; datasheet A11873-2).
How should I adapt the localisation check for multiplex IF?
For multiplex IF, pair SEMA4F with a marker identifying the cell type or structure under study, and inspect overlap within segmented cells rather than relying on adjacent bright pixels (standard IF practice). Retinal nerve fibers are a supported tissue-IHC reference, while HPA supplies no cell lines with ICC/IF images for this target (HPA tissue IHC; HPA subcellular). Choose fluorophores after checking tissue autofluorescence in an unstained section, and reserve the cleaner channel for the weaker signal (standard IF practice). Set permeabilisation according to whether the antibody recognises the extracellular 35–659 region or cytoplasmic 681–770 region; its epitope side is not supplied (UniProt O95754 topology; datasheet A11873-2).
How do I reduce background without losing specific SEMA4F signal?
Use a no-primary section to identify detection background, and include a peroxidase block before HRP/DAB development (standard chromogenic IHC practice). The catalog image used 10% goat serum blocking, 2 μg/ml primary overnight at 4°C, and a goat anti-rabbit secondary for 30 minutes at 37°C (datasheet A11873-2). If background persists, titrate primary concentration and DAB development while holding retrieval and section handling constant (standard IHC practice). HPA reports presumed off-target binding in its tissue-IHC assessment, so compare any new staining pattern with cellular morphology and an independent control before calling it SEMA4F (HPA tissue IHC).
What should I measure when comparing SEMA4F across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define regions and cell types before scoring, then report either an H-score, percentage of positive cells, or positive fiber area per mm², as appropriate to the observed pattern (standard IHC practice). Normalise cell counts to the number of evaluable cells and fiber measurements to viable tissue area, using the same threshold and DAB development conditions across sections (standard IHC practice). Keep retinal nerve fibers separate from other compartments because the documented medium signal is in the nerve fiber layer (HPA tissue IHC). Record background and excluded damaged areas, and report staining intensity separately from area when a single score would hide a pattern change (standard IHC practice).
How can I distinguish true SEMA4F signal from artefact?
Check whether signal follows membranes or cell processes, consistent with SEMA4F’s membrane, dendritic, and postsynaptic annotations (UniProt O95754 subcellular location). In retina, compare the nerve fiber layer with nearby structures; medium staining is reported there, whereas several sampled glandular and other cell populations were not detected in the HPA tissue-IHC panel (HPA tissue IHC). Treat staining at section edges, in necrotic areas, or in a no-primary control as suspect, and check peroxidase blocking when DAB appears broadly deposited (standard chromogenic IHC practice). HPA’s approved assessment explicitly notes presumed off-target binding, so a plausible compartment alone does not establish specificity (HPA tissue IHC).
Boster reagents
Best SEMA4F / Semaphorin-4F IHC Antibodies
The IHC-validated antibody has images from human paraffin sections and IF images from U2OS cells (catalog image captions); listed reactivity includes human, mouse and rat (catalog reactivity).
Real IHC data
Anti-SEMA4F Antibody ®
Cat # A11873-2
A11873-2 has IHC images from human paraffin sections of colon adenocarcinoma, larynx squamous cell carcinoma, liver cancer and lung adenocarcinoma (catalog IHC image captions). A11873-2 also has an IF image from U2OS cells (catalog IF image caption); human, mouse and rat are listed as reactive species (catalog reactivity).
Which to pick: Choose A11873-2 for chromogenic IHC on paraffin sections: its IHC captions document EDTA retrieval at pH 8.0, 2 μg/ml primary antibody and DAB detection; the fixative is unreported (catalog IHC image captions). For IF/ICC, A11873-2 has an image from U2OS cells at 5 μg/ml (catalog IF image caption). It is also the listed choice for mouse or rat samples based on reactivity, although the supplied images show human tissue and U2OS cells; clonality is unreported (catalog reactivity; catalog image captions; catalog clone field).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O95754 (SEM4F_HUMAN, Semaphorin-4F).
- Human Protein Atlas. SEMA4F tissue IHC expression (reliability: Approved).
- Human Protein Atlas. SEMA4F subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. SEMA4F antibody validation summary (1 antibodies).
- Establishing Molecular Subgroups of CD8+ T Cell-Associated Genes in the Ovarian Cancer Tumour Microenvironment and Predicting the Immunotherapy Response. Biomedicines 2023 — PMC10525231.
- PubMed PMID:10051670 — UniProt-cited evidence.
- PubMed PMID:11230166 — UniProt-cited evidence.
- PubMed PMID:16303743 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for SEMA4F (Semaphorin-4F). Expected localisation Retinal nerve fibers (HPA tissue IHC); membrane expected (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A11873-2). Validated IHC antibody: A11873-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Retina (Nerve fiber layer); negative Caudate. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon adenocarcinoma tissue; fixative not specified (datasheet A11873-2); 2. Fixation: Image fixative and duration unreported (datasheet A11873-2); 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 A11873-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A11873-2); 8. Primary antibody: Rabbit anti-SEMA4F, 2-5 μg/ml (datasheet A11873-2); 9. Primary incubation: Overnight at 4 °C (datasheet A11873-2). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A11873-2); Caveat: Presumed off-target staining was disregarded (HPA tissue IHC); Isoform / epitope: 2 isoforms; check whether the epitope is extracellular or cytoplasmic (UniProt); Regulation: Regulation not annotated (UniProt). Evidence: UniProt O95754, HPA tissue IHC · Approved, HPA ICC-IF · Membrane, Datasheet A11873-2, PMC10525231. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/SEMA4F/immunohistochemistry-ihc-guide.