SEMA6A / Semaphorin-6A · IHC design guide

Design Immunohistochemistry for SEMA6A

Plan paraffin-section SEMA6A IHC with the catalog antibody at 0.5–1 μg/ml (datasheet A06967-1). Use the observed granular cytoplasmic pattern and tissue controls to interpret staining alongside SEMA6A’s membrane topology (HPA tissue IHC; UniProt).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for SEMA6A (IHC for SEMA6A): expected localisation Granular cytoplasm (HPA tissue IHC); cell membrane (UniProt), antibody A06967-1, validated IHC image, and IHC protocol steps — Printable **SEMA6A** IHC protocol sheet — expected localisation Granular cytoplasm (HPA tissue IHC); cell membrane (UniProt), antibody A06967-1, controls and protocol steps. Open the full SEMA6A IHC guide →

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SEMA6A 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 (HPA tissue IHC); cell membrane (UniProt)
	Staining pattern	Granular cytoplasmic staining in glandular and neuronal cells (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A06967-1)
	Positive control ⓘ	Adrenal gland+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 sections. (standard IHC practice; not target-specific)
	Caveat	Tissue staining and RNA show only medium consistency (HPA tissue IHC)
	Regulation	Adrenal gland and brain: tissue-enhanced RNA (HPA tissue RNA)
	Isoform / epitope	2 isoforms; map the epitope to the extracellular or cytoplasmic region (UniProt)

Section 1

Recommended SEMA6A IHC & IF Protocols

The catalog antibody uses EDTA pH 8.0 heat retrieval (datasheet A06967-1). Published IHC methods for HCC and melanoma provide additional protocol details (PMC12001175; PMC9016967).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human rectal cancer tissue; fixative not specified (datasheet A06967-1)
Fixation	Image fixative and duration unreported (datasheet A06967-1); 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 A06967-1); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A06967-1)
Primary antibody	Rabbit anti-SEMA6A, 0.5-1μg/ml (datasheet A06967-1)
Primary incubation	Overnight at 4 °C (datasheet A06967-1)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A06967-1)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	SEMA6A-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression with a granular pattern. No signal in the no-primary control.

💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet A06967-1). The melanoma method used citrate pH 6 with a different primary antibody (PMC9016967).

Section 2

What Is the Expected SEMA6A Staining Pattern?

SEMA6A is a cell-membrane protein with an extracellular region, one transmembrane segment, and a cytoplasmic tail (UniProt Q9H2E6 topology). In paraffin-section IHC, HPA describes a general granular cytoplasmic pattern, with high staining in several glandular, hematopoietic, respiratory epithelial, and neural cell populations (HPA tissue IHC). HPA rates the tissue staining Approved, with medium consistency between antibody staining and RNA expression (HPA tissue IHC).

What am I looking at on my slide?
Granular cytoplasmic staining in adrenal glandular cells or cerebellar Purkinje cells.	This matches HPA's reported pattern and high staining in those cells (HPA tissue IHC). A continuous membrane outline is not required by the reported IHC pattern, although membrane localization is consistent with SEMA6A topology (UniProt Q9H2E6 topology).
Strong nuclear-only staining in a paraffin section, without the expected granular cytoplasmic pattern.	Treat this IHC result as discordant and investigate staining specificity or an imaging artefact (HPA tissue IHC; UniProt Q9H2E6 topology). HPA separately reports nucleoplasmic ICC-IF localization, so nuclear signal across methods cannot be dismissed categorically (HPA subcellular ICC-IF).
Strong staining confined to myocytes, chondrocytes, or adipocytes while expected high-staining cells are unstained.	These cell types are listed as low-staining comparators; the distribution raises concern for cross-reactivity or endogenous chromogenic activity (HPA tissue IHC; general IHC practice). Low staining is not proof of absence (HPA tissue IHC).
Uniform color over cells and surrounding tissue, obscuring cell boundaries.	This is difficult to score as SEMA6A because HPA describes a cellular granular pattern (HPA tissue IHC). Assess nonspecific background and the chromogenic detection controls before assigning positivity (general IHC practice).
No detectable staining in a suitable section containing HPA high-staining cells.	Check assay performance before interpreting other sections as negative (HPA tissue IHC; general IHC practice). HPA's Approved rating has medium staining–RNA consistency, so one discordant specimen does not establish target absence (HPA tissue IHC).

💡Expected SEMA6A appearanceA convincing positive paraffin-section IHC result shows granular cytoplasmic staining in an HPA high-staining cell population, such as adrenal glandular cells or cerebellar Purkinje cells; diffuse tissue-wide color or isolated nuclear staining needs investigation (HPA tissue IHC; UniProt Q9H2E6 topology).

How each factor affects the staining
Epitope position and topology	SEMA6A has an extracellular region at residues 19–649, a transmembrane segment at 650–670, and a cytoplasmic region at 671–1030 (UniProt Q9H2E6 topology). An antibody's mapped epitope would help interpret which portion is detected; no epitope position is supplied here.
Processing and glycosylation	UniProt lists a signal peptide at residues 1–18, a chain at 19–1030, and six glycosylation sites (UniProt Q9H2E6 processing/PTMs). These annotations do not establish shedding or a target-specific antigen-retrieval requirement.
Tissue choice and evidence strength	HPA reports high staining in adrenal glandular cells, bone-marrow hematopoietic cells, and several neural populations, while listing myocytes, chondrocytes, and adipocytes as low (HPA tissue IHC). Use the stated cell type when judging a section; HPA rates the tissue IHC Approved with medium staining–RNA consistency (HPA tissue IHC).
Antibody validation	HPA lists HPA031265 as IHC Approved; the other listed antibodies have no IHC status in this payload (HPA antibody validation). An Approved result supports interpretation of its reported pattern but does not establish identical performance for another antibody (HPA antibody validation; general IHC practice).
IF/ICC Q: Should nuclear IF signal define the expected IHC result?	HPA reports approved nucleoplasmic, nuclear-body, and intermediate-filament ICC-IF locations, while its tissue IHC profile is granular cytoplasmic (HPA subcellular ICC-IF; HPA tissue IHC). The listed antibodies have ICC status Uncertain, so assess IF findings in their own assay context (HPA antibody validation).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
No staining in an HPA high-staining cell population.	The assay may have failed, or that specimen may differ from the HPA observation (HPA tissue IHC; general IHC practice).	Check the section and positive control, then review the IHC antibody conditions and chromogenic detection steps (general IHC practice). Avoid calling the specimen target-negative until controls work.
Granular staining is present but too faint to score.	Weak detection or counterstain that overwhelms the chromogen can limit visibility (general IHC practice). HPA's high category does not specify an intensity for every specimen (HPA tissue IHC).	Compare with an HPA high-staining cell population and inspect control performance; adjust validated detection or counterstaining conditions if needed (HPA tissue IHC; general IHC practice).
Diffuse color covers tissue and blank areas.	Nonspecific background or chromogenic detection activity may obscure the cellular pattern (general IHC practice).	Inspect negative and detection controls, blocking, washing, and primary-antibody concentration before scoring SEMA6A (general IHC practice). Look for HPA's granular cellular pattern (HPA tissue IHC).
Signal appears nuclear-only in IHC.	The result conflicts with HPA's granular tissue-IHC profile, though HPA reports nucleoplasmic localization in ICC-IF (HPA tissue IHC; HPA subcellular ICC-IF).	Verify cell boundaries and counterstain, examine controls, and compare a known high-staining tissue section under the same IHC conditions (HPA tissue IHC; general IHC practice).
Strong color appears mainly in HPA low-staining cell types.	Cross-reactivity or endogenous detection activity is possible; HPA lists myocytes, chondrocytes, and adipocytes as low, not negative (HPA tissue IHC; general IHC practice).	Compare the signal with HPA high-staining cells and inspect negative and detection controls before assigning specificity (HPA tissue IHC; general IHC practice).
Results differ between antibodies or sections.	The supplied HPA antibody records do not give every antibody the same IHC validation status, and tissue-IHC staining has medium consistency with RNA data (HPA antibody validation; HPA tissue IHC).	Record the antibody identity and control results, then compare the same cell types and staining compartments across sections (general IHC practice; HPA tissue IHC).

Source: UniProt Q9H2E6 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing SEMA6A staining

IHC analysis of Sema6A using anti-Sema6A antibody (A06967-1). Sema6A was detected in paraffin-embedded section of human rectal cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-Sema6A Antibody (A06967-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.

A06967-1 · IHCIHC analysis of Sema6A using anti-Sema6A antibody (A06967-1). Sema6A was detected in paraffin-embedded section of human rectal cancer tissue

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000092421-SEMA6A/tissue/cerebral+cortex#imid_7782672

HPA · CerebellumHuman Protein Atlas IHC, Cerebellum. Source: https://www.proteinatlas.org/ENSG00000092421-SEMA6A/tissue/cerebellum#imid_7782683

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000092421-SEMA6A/tissue/colon#imid_7782758

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000092421-SEMA6A/tissue/liver#imid_7782759

HPA · ICC-IF HAP1Human Protein Atlas immunofluorescence, HAP1, antibody HPA031264; green = SEMA6A, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for SEMA6A IHC & IF

🧪Start with bronchus and expect staining in respiratory epithelial cells (High; HPA: bronchus). HPA detects SEMA6A in all 45 scored tissues, so use no-primary and isotype controls for the negative; cells without visible signal on the bronchus slide can serve as internal background references, but should not be designated SEMA6A-negative (HPA: no negative tissues listed).

Positive control tissue: Adrenal gland (Glandular cells, HPA High)

Negative control tissue: None in HPA: SEMA6A 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 SEMA6A in HAP1, RT-4, Hep-G2, U2OS, NIH 3T3, with annotated localisation: Nucleoplasm (approved) (HPA subcellular).

Technical controls: Run a no-primary, secondary-only control and a concentration-matched rabbit IgG isotype control alongside a SEMA6A knockout specimen as a biological negative (caption: rabbit primary antibody). For ABC/DAB detection, quench endogenous peroxidase and check for endogenous biotin signal in the bronchus section (caption: biotinylated secondary, streptavidin–biotin complex and DAB).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected paraffin-section caption does not report a fixative (caption: fixative not stated). The caption used heat-mediated retrieval in EDTA at pH 8.0; whether SEMA6A staining depends on that retrieval condition is unreported (caption: rectal cancer tissue IHC). Neither frozen sections nor IF can be called easier from this evidence; in bronchus, inspect mucus and inflammatory cells for nonspecific chromogen signal when interpreting epithelial staining (HPA: High in respiratory epithelial cells; caption: DAB detection).

HPA tissue IHC evidence for SEMA6A

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.

Positive expression · recommended positive controls

Tissue	Cell type	Level	Evidence	Source
Adrenal gland	Glandular cells	High	Protein (IHC)	HPA →
Appendix	Glandular cells	High	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	High	Protein (IHC)	HPA →
Breast	Glandular cells	High	Protein (IHC)	HPA →
Bronchus	Respiratory epithelial cells	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
None in HPA: SEMA6A is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

Section 3

Advanced SEMA6A IHC Tips

Troubleshoot SEMA6A staining by checking retrieval, compartment, cell identity and matched controls before interpreting signal intensity.

Which retrieval condition should I start with for SEMA6A paraffin-section IHC?

Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A06967-1). The selected tissue image used this retrieval before overnight incubation at 4°C with 1 μg/ml antibody (caption A06967-1). If staining is weak, compare retrieval duration on adjacent sections while keeping antibody concentration, detection and development consistent (standard IHC practice). Check tissue preservation and a positive control before concluding that retrieval failed (standard IHC practice). Excessive retrieval can damage morphology, making granular cytoplasmic staining harder to judge against the reported tissue pattern (standard IHC practice; HPA tissue IHC).

How should I troubleshoot fixation-related loss of SEMA6A staining?

The selected image identifies a paraffin-embedded section but does not state its fixative, so target-specific fixation sensitivity is unknown (caption A06967-1). Record the actual fixative and processing history for each specimen, and compare sections processed under matched conditions when troubleshooting weak signal (standard IHC practice). Apply the documented EDTA pH 8.0 retrieval and 1 μg/ml primary concentration before changing other variables (datasheet A06967-1; caption A06967-1). If signal differs across processing batches, assess morphology and control tissue alongside staining, because those observations alone cannot establish a SEMA6A-specific fixation effect (standard IHC practice).

How should membrane and cytoplasmic SEMA6A staining be reconciled?

SEMA6A is annotated as a cell-membrane protein with an extracellular region at residues 19–649 and a cytoplasmic region at 671–1030 (UniProt Q9H2E6 topology). Tissue IHC also reports general granular cytoplasmic staining, with only medium consistency between staining and RNA expression (HPA tissue IHC). Score membrane-associated and granular cytoplasmic patterns separately, using the same criteria across sections (standard IHC practice). Diffuse nuclear staining deserves additional scrutiny because it differs from the membrane annotation, although approved ICC/IF data report nucleoplasmic localisation (UniProt Q9H2E6 subcellular; HPA subcellular). Resolve disagreement with compartment controls and an independently validated antibody or orthogonal assay (standard IHC practice).

Could isoforms or epitope accessibility explain discordant SEMA6A IHC?

SEMA6A has 2 annotated isoforms, but the supplied evidence does not map the catalog antibody’s epitope to either one (UniProt Q9H2E6 isoforms; caption A06967-1). Its transmembrane segment spans residues 650–670, separating extracellular and cytoplasmic regions that may present different accessibility in sections (UniProt Q9H2E6 topology; standard IHC practice). The protein also has 6 annotated glycosylation sites, which is relevant when interpreting potential epitope masking, without establishing an effect for this antibody (UniProt Q9H2E6 glycosylation). Compare documented epitope information with isoform sequences if available, and test any proposed explanation against matched positive controls and an independent reagent (standard IHC practice).

How should I investigate SEMA6A signal by multiplex IF?

Use IF/ICC as a separate validation experiment, since the selected antibody evidence here describes paraffin-section chromogenic IHC (caption A06967-1). Pair SEMA6A with a marker for an expected cell population, such as Purkinje cells, and verify cell identity before interpreting overlap (HPA tissue IHC). Choose well-separated fluorophores and consider a far-red channel when tissue autofluorescence obscures shorter wavelengths (standard IF practice). Set permeabilisation according to the mapped epitope: an extracellular epitope may be accessible without permeabilisation, whereas a cytoplasmic epitope generally requires membrane access (UniProt Q9H2E6 topology; standard IF practice). Include single-stain and secondary-only controls to assess bleed-through and nonspecific fluorescence (standard IF practice).

What should I check when SEMA6A DAB staining appears widespread?

Check a no-primary control for secondary-reagent background and endogenous peroxidase signal before attributing widespread DAB deposit to SEMA6A (standard chromogenic IHC practice). The selected image used 10% goat-serum blocking, biotinylated secondary antibody, streptavidin-biotin detection and DAB (caption A06967-1). Include an endogenous peroxidase block and assess possible endogenous biotin background when using that detection system (standard chromogenic IHC practice). Keep chromogen development and section thickness consistent across comparisons, and inspect edges, folds and necrotic areas separately (standard IHC practice). A granular cytoplasmic pattern is reported in tissue IHC, so uniform staining across unrelated structures needs control-based verification (HPA tissue IHC; standard IHC practice).

How can I quantify SEMA6A staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and scoring compartment before analysis, then apply those definitions consistently to every section (standard IHC practice). For cellular staining, record the percentage of positive cells and an H-score based on staining intensity; for spatially restricted cells, density per mm² may be more useful (standard IHC practice). Normalise counts to viable tissue area or the number of evaluable cells in the same compartment, and exclude folds and necrosis using preset rules (standard IHC practice). Report membrane-associated and granular cytoplasmic signal separately because both the membrane annotation and tissue pattern are relevant to interpretation (UniProt Q9H2E6 subcellular; HPA tissue IHC).

Which findings support genuine SEMA6A staining rather than artefact?

A credible result follows reproducible cell and compartment patterns, with control sections showing acceptably low background (standard IHC practice). High staining has been reported in cerebellar Purkinje cells and caudate neuronal cells, while skeletal-muscle myocytes are listed as low (HPA tissue IHC). Interpret those comparisons cautiously because the tissue IHC profile has medium consistency with RNA expression (HPA tissue IHC). Membrane-associated signal fits the annotated protein topology, while granular cytoplasmic staining matches the reported tissue pattern (UniProt Q9H2E6 topology; HPA tissue IHC). Treat edge accentuation, necrotic deposits, unexplained nuclear-only DAB and signal reproduced without primary antibody as prompts to investigate artefact (standard IHC practice).

Boster reagents

Best SEMA6A / Semaphorin-6A IHC Antibodies

The catalog antibody A06967-1 has real IHC data from a human rectal cancer paraffin section (A06967-1 IHC caption); its listed reactivity is human, mouse and rat (catalog: reactivity).

Real IHC data

Anti-Sema6A Antibody ®

Cat # A06967-1

View product →

A06967-1 will render with IHC data from a human rectal cancer paraffin section (A06967-1 IHC caption). The catalog lists IHC among its applications and human, mouse and rat reactivity; it provides no IF image or IF dilution (catalog: applications, reactivity, IF fields).

Which to pick: Choose A06967-1 for paraffin-section tissue IHC: its own caption documents heat retrieval in EDTA at pH 8.0 and 1 μg/ml primary antibody; the fixative is unreported (A06967-1 IHC caption). No IF/ICC-validated SKU is supplied, so there is no supported IF/ICC pick (catalog: applications, IF fields). A06967-1 is the only listed cross-species candidate based on human, mouse and rat reactivity, but its IHC dilution is specified for human samples (catalog: reactivity, dilution_raw).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

UniProt Consortium. UniProt entry Q9H2E6 (SEM6A_HUMAN, Semaphorin-6A).
Human Protein Atlas. SEMA6A tissue IHC expression (reliability: Approved).
Human Protein Atlas. SEMA6A subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the nuclear bodies and intermediate filaments..
Human Protein Atlas. SEMA6A antibody validation summary (3 antibodies).
Semaphorin-Plexin signaling influences early ventral telencephalic development and thalamocortical axon guidance. Neural development 2017 — PMC5402653.
Long noncoding RNA semaphorin 6A-antisense RNA 1 reduces hepatocellular carcinoma by promoting semaphorin 6A mRNA degradation. World journal of gastroenterology 2025 — PMC12001175.
Semaphorin-6A controls guidance of corticospinal tract axons at multiple choice points. Neural development 2008 — PMC2647909.
SEMA6A/RhoA/YAP axis mediates tumor-stroma interactions and prevents response to dual BRAF/MEK inhibition in BRAF-mutant melanoma. Journal of experimental & clinical cancer research : CR 2022 — PMC9016967.
PubMed PMID:10993894 — UniProt-cited evidence.
PubMed PMID:10718198 — UniProt-cited evidence.
PubMed PMID:15372022 — UniProt-cited evidence.