SEMA6A / Semaphorin-6A · IHC design guide

Design Immunohistochemistry for SEMA6A

Plan SEMA6A paraffin-section IHC around the observed granular cytoplasmic pattern (HPA tissue IHC) and the annotated membrane topology (UniProt). This guide covers staining controls, interpretation, and the catalog antibody’s IHC conditions (datasheet A06967-1).

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

Navigate to: Protocols Staining pattern Controls Antibodies

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 observed (HPA tissue IHC); cell membrane annotated (UniProt)
	Staining pattern	Granular cytoplasmic staining across multiple cell types (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). Selected-image fixative and duration unreported (datasheet A06967-1); verify before use.
	Caveat	Granular tissue staining may differ from membrane expectation (HPA tissue IHC) (UniProt)
	Regulation	Regulation not annotated (UniProt)
	Isoform / epitope	2 isoforms; check extracellular versus cytoplasmic epitope (UniProt)

Section 1

Recommended SEMA6A IHC & IF Protocols

The catalog antibody’s paraffin IHC protocol (datasheet: A06967-1) is paired with published SEMA6A staining methods from liver, mouse developmental sections, and melanoma (PMC12001175; PMC2647909; 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 heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet: A06967-1); published methods also used citrate buffers (PMC2647909; PMC9016967).

Section 2

What Is the Expected SEMA6A Staining Pattern?

SEMA6A is a cell-membrane protein with an extracellular region and cytoplasmic tail (UniProt Q9H2E6 topology). In tissue IHC, HPA reports predominantly granular cytoplasmic staining, including high staining in adrenal glandular cells, cerebellar Purkinje cells and cerebral-cortex neuropil (HPA tissue IHC). Treat this observed pattern as a guide: HPA rates the tissue staining Approved, with medium consistency between antibody staining and RNA expression (HPA tissue IHC reliability).

What am I looking at on my slide?
Granular cytoplasmic staining in adrenal glandular cells or cerebellar Purkinje cells (HPA tissue IHC).	This matches the reported tissue pattern and high staining level (HPA tissue IHC). Score the named cell population and compare its staining with nearby tissue on the same section (general IHC practice).
A membrane-edge signal accompanies granular cytoplasmic staining in the expected cells.	A membrane-associated component is biologically plausible because SEMA6A has one transmembrane segment (UniProt Q9H2E6 topology). The granular cytoplasmic pattern remains the tissue-IHC reference; membrane-only staining needs further validation (HPA tissue IHC).
Strong nuclear-only staining replaces the expected granular cytoplasmic pattern.	For paraffin IHC, investigate an off-pattern signal or artefact against the HPA tissue-IHC profile. Do not use nuclear ICC-IF localisation to validate this IHC result: HPA reports nucleoplasm in ICC-IF, while its listed antibodies have Uncertain ICC status (HPA subcellular; HPA antibodies).
Intense staining appears chiefly in an unexpected cell population.	Check cell identity against the HPA tissue record; cross-reactivity or endogenous detection activity are possible IHC explanations (HPA tissue IHC; general IHC practice). HPA supplies no formally negative tissue here, so unexpected staining alone cannot prove non-specificity (HPA tissue IHC).
Broad, diffuse colour obscures cell boundaries and the granular pattern.	Interpretability is low when background prevents recognition of the reported pattern (HPA tissue IHC). Background from detection reagents, incomplete blocking or excess primary antibody is a general IHC possibility, not a demonstrated SEMA6A-specific effect (general IHC practice).

💡Expected SEMA6A appearanceCall a result convincing when granular cytoplasmic staining is distinct in HPA high-staining cells, such as adrenal glandular cells or Purkinje cells; diffuse tissue-wide colour or nuclear-only IHC is off-pattern (HPA tissue IHC).

How each factor affects the staining
Compartment and epitope location	SEMA6A spans the membrane at residues 650–670, with extracellular residues 19–649 and cytoplasmic residues 671–1030 (UniProt Q9H2E6 topology). The supplied record does not locate the antibody epitope, so it cannot predict whether a given stain will emphasize the cell edge or intracellular material.
Tissue and cell selection	HPA reports high staining in adrenal glandular cells, bone-marrow hematopoietic cells, cerebellar Purkinje cells and cerebral-cortex neuropil; skeletal-muscle myocytes, chondrocytes and adipocytes are listed as low (HPA tissue IHC). Low staining is not a validated negative control.
Antibody validation strength	HPA031265 is IHC Approved; the other listed antibodies have no IHC status in this payload (HPA antibodies). The tissue profile is Approved with medium RNA–staining consistency, so compare morphology and controls before assigning weak or unusual signal to SEMA6A (HPA tissue IHC reliability; general IHC practice).
Molecular features	UniProt lists 2 isoforms, a signal peptide at residues 1–18 and 6 glycosylation sites (UniProt Q9H2E6). These features alone do not establish epitope recognition, shedding, retrieval requirements or a fixation effect for the catalog antibody.
Antigen retrieval	Retrieval conditions are an assay variable in paraffin IHC (general IHC practice). Neither the HPA tissue pattern nor UniProt topology establishes SEMA6A-specific retrieval needs or fixation sensitivity; judge retrieval changes using staining pattern and controls.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
No staining in adrenal glandular cells or cerebellar Purkinje cells (HPA tissue IHC).	The tissue may be unsuitable, or an IHC workflow step may have failed; absence of signal alone does not identify the cause (general IHC practice).	Verify the named cells are present, then check the primary antibody, retrieval, detection reagents and a working positive-control section (general IHC practice).
Only weak staining is visible in skeletal-muscle myocytes, chondrocytes or adipocytes (HPA tissue IHC).	HPA lists these cell populations as low, so they provide limited sensitivity checks and are not formal negatives (HPA tissue IHC).	Assess performance in an HPA high-staining population on a suitable section before changing the assay (HPA tissue IHC; general IHC practice).
Colour covers most cells, including populations outside the expected pattern.	Primary-antibody background, non-specific secondary binding or endogenous detection activity are possible general IHC causes (general IHC practice).	Compare a primary-omission control, review blocking and detection controls, and adjust antibody concentration only against an interpretable positive control (general IHC practice).
Nuclear-only IHC signal dominates the section.	This conflicts with the HPA granular tissue-IHC profile, although HPA separately reports approved nucleoplasmic localisation in ICC-IF (HPA tissue IHC; HPA subcellular).	Review morphology and controls; require independent IHC support before treating nuclear colour as the expected paraffin-section pattern (general IHC practice).
Signal is present, but no granular cytoplasmic detail can be resolved.	Excess colour development or diffuse background can conceal the pattern used for interpretation (HPA tissue IHC; general IHC practice).	Review development and counterstain, then compare the same expected cell population under conditions that preserve cellular detail (general IHC practice).
Does the reported nucleoplasmic ICC-IF signal define the expected IHC result?	No: ICC-IF and paraffin IHC are separate assays; HPA reports nucleoplasm in ICC-IF but marks the listed antibodies Uncertain for ICC (HPA subcellular; HPA antibodies).	Interpret paraffin IHC against the granular tissue profile. Assess any IF/ICC result with assay-specific controls in its own guide (HPA tissue IHC; general IF/ICC practice).

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

🧪Run breast first: its glandular cells should stain (HPA: High in breast glandular cells). HPA detects SEMA6A in all 45 scored tissues, so there is no HPA negative tissue; use no-primary and isotype controls, and assess adjacent stromal cells for staining above background without assuming they are SEMA6A-negative (HPA: no negative tissue 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: Include a no-primary, secondary-only slide, a concentration-matched rabbit IgG isotype control, and a SEMA6A knockout specimen as a biological negative (caption: rabbit primary antibody; standard IHC controls). Quench endogenous peroxidase and check for endogenous biotin signal in breast tissue when using the caption’s biotin-based DAB detection (caption: biotinylated secondary, streptavidin–biotin complex, DAB).

⚠️Feasibility: Paraffin-section staining is documented with heat retrieval in EDTA at pH 8.0, but whether retrieval is required has not been established (selected-SKU tissue-IHC caption). A target-specific fixation window or fixation effect is unreported, and the caption does not state the fixative (selected-SKU tissue-IHC caption). There is no supplied comparison showing that frozen sections or IF are easier; assess glandular staining against stromal background and the biotin-based detection controls (HPA: High in breast glandular cells; selected-SKU tissue-IHC caption).

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 in paraffin sections by checking retrieval, compartment, cell type and assay controls before assigning biological meaning.

What retrieval should I use if SEMA6A staining is weak in paraffin sections?

Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A06967-1). The selected antibody stained a paraffin-embedded human rectal cancer section after this retrieval, followed by 1 μg/ml primary antibody overnight at 4°C (datasheet A06967-1). If staining remains weak, vary heating time on matched sections while keeping the buffer and antibody concentration fixed (standard IHC practice). Compare signal in intact tissue with a no-primary control, because stronger DAB deposition alone does not establish specificity (standard IHC practice). Record heating duration and cooling conditions so any gain can be reproduced (standard IHC practice).

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

Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative (datasheet A06967-1). Record the actual fixative and fixation duration for each specimen before comparing staining across blocks (standard IHC practice). If matched tissue is available, compare sections processed with different documented fixation durations using the same pH 8.0 EDTA retrieval (datasheet A06967-1; standard IHC practice). Keep section thickness, antibody concentration and DAB development consistent, then judge preserved cell morphology alongside signal (standard IHC practice). Do not assign weak staining to fixation without that comparison and appropriate positive and no-primary controls (standard IHC practice).

Should I expect membrane staining or granular cytoplasmic staining in SEMA6A IHC?

Interpret both patterns cautiously: 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 instead reports general granular cytoplasmic expression, with approved staining of medium RNA concordance (HPA tissue IHC). Ask whether staining outlines cells, forms intracellular granules, or tracks section edges, and score those patterns separately (standard IHC practice). A diffuse nuclear DAB signal needs independent verification; approved nucleoplasmic localisation comes from ICC/IF data, not this tissue IHC assay (HPA subcellular). Confirm any proposed compartment with antibody specificity controls before interpreting it biologically (standard IHC practice).

Could SEMA6A isoforms or epitope location explain discordant staining?

SEMA6A has 2 annotated isoforms, so first establish which sequence region the antibody recognises before comparing specimens (UniProt Q9H2E6 isoforms; standard IHC practice). Its extracellular segment spans residues 19–649, its membrane segment 650–670, and its cytoplasmic segment 671–1030 (UniProt Q9H2E6 topology). Six annotated glycosylation sites occur in the extracellular region, while phosphoserines are reported at 698 and 952 (UniProt Q9H2E6 PTMs). These annotations identify plausible epitope-context questions; they do not establish interference with this antibody (UniProt Q9H2E6; standard IHC interpretation). Compare epitope coverage and matched staining controls before attributing discordance to an isoform or modification (standard IHC practice).

How can I check an IHC pattern with multiplex SEMA6A immunofluorescence?

Use multiplex IF as a separate validation experiment, since the selected antibody caption documents paraffin-section chromogenic IHC, with no IF conditions supplied (datasheet A06967-1). Pair SEMA6A with a validated marker for the cell population under study; Purkinje cells or adrenal glandular cells offer documented high tissue IHC signal to investigate (HPA tissue IHC). Choose spectrally separated fluorophores after measuring tissue autofluorescence, and include single-stain and no-primary controls (standard IF practice). If the antibody recognises residues 19–649, assess surface access before permeabilisation; a cytoplasmic epitope at 671–1030 requires membrane access (UniProt Q9H2E6 topology; standard IF practice). Compare compartment patterns with chromogenic sections without treating ICC/IF nucleoplasmic localisation as tissue-IHC confirmation (HPA subcellular; standard assay interpretation).

What should I check when SEMA6A DAB staining looks diffuse?

First inspect a no-primary section for secondary-reagent binding and endogenous enzyme signal, using the same DAB development interval (standard IHC practice). The selected workflow used 10% goat serum, biotinylated goat anti-rabbit secondary antibody, an avidin–biotin complex and DAB (datasheet A06967-1). Check peroxidase blocking and, where relevant, endogenous biotin with appropriate controls before assigning diffuse brown signal to SEMA6A (standard IHC practice). Compare background in tissue edges, folds and damaged regions with intact cells on the same slide (standard IHC practice). If background persists, titrate primary antibody around the documented 1 μg/ml condition while holding detection and imaging constant (datasheet A06967-1; standard IHC practice).

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

Define the scored compartment and cell population before analysis, because membrane localisation and granular cytoplasmic tissue staining are both reported in different evidence sources (UniProt Q9H2E6 topology; HPA tissue IHC). For cellular DAB staining, report percent positive cells and an H-score based on prespecified intensity bins (standard IHC practice). For a spatially restricted population, report positive-cell density per mm² of viable tissue instead (standard IHC practice). Normalise counts to the number of eligible cells or viable tissue area, and apply identical thresholds across sections (standard IHC practice). Exclude folds, necrosis and edge artefacts, and keep cell-type strata separate during comparison (standard IHC practice).

When is a SEMA6A-positive IHC result biologically credible?

A credible result shows a repeatable cellular pattern in intact tissue and exceeds signal in a matched no-primary control (standard IHC practice). Membrane staining fits the annotated topology, while granular cytoplasmic staining has tissue-IHC support; neither pattern alone proves antibody specificity (UniProt Q9H2E6 topology; HPA tissue IHC; standard IHC interpretation). Check whether signal occurs in documented high-staining populations, such as cerebellar Purkinje cells or adrenal glandular cells, when those tissues are available (HPA tissue IHC). Treat isolated nuclear signal cautiously because approved nucleoplasmic localisation derives from ICC/IF observations (HPA subcellular). Discount staining confined to edges or necrosis, and exclude endogenous peroxidase or biotin signal with detection controls (standard IHC practice).

Boster reagents

Best SEMA6A / Semaphorin-6A IHC Antibodies

A06967-1 has real IHC data from a paraffin-embedded human rectal cancer section (catalog figure). Its listed reactivity includes human, mouse, and rat (catalog reactivity).

Real IHC data

Anti-Sema6A Antibody ®

Cat # A06967-1

View product →

A06967-1 is listed for IHC and for human, mouse, and rat reactivity (catalog applications/reactivity). Its IHC figure shows chromogenic detection in a paraffin-embedded human rectal cancer section after EDTA pH 8 retrieval (A06967-1 figure caption).

Which to pick: Choose A06967-1 for paraffin-section tissue IHC at the listed 0.5–1 μg/ml; its figure used 1 μg/ml, and the fixative is unreported (catalog IHC dilution; A06967-1 figure caption). No IF/ICC application or IF figure is supplied for this SKU, so there is no supported IF/ICC pick here (catalog applications; catalog IF images). For mouse or rat samples, A06967-1 lists reactivity, but its supplied IHC figure shows human tissue only (catalog reactivity; A06967-1 figure caption).

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.