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SYNE3 / Nesprin-3 · IHC design guide
Design Immunohistochemistry for SYNE3
Plan SYNE3 chromogenic IHC on paraffin sections with the catalog antibody's 2–5 μg/ml range (datasheet A13164-2). Compare staining with the reported nuclear pattern and high signal in testis spermatogonia (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
SYNE3 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 | Nuclear staining in tissue (HPA tissue IHC); outer nuclear membrane location (UniProt) | |
| Staining pattern | Nuclear staining in several tissues; high in spermatogonia (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A13164-2) | |
| Positive control | Testis | |
| Negative control | Adipose tissue+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 A13164-2) | |
| Caveat | Bone marrow RNA is enhanced, but hematopoietic IHC is undetected (HPA tissue IHC) | |
| Regulation | Tissue-enhanced RNA in bone marrow (HPA tissue RNA) | |
| Isoform / epitope | 3 isoforms; epitope differences unspecified (UniProt) |
Section 1
Recommended SYNE3 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet A13164-2) with a published SYNE3 IHC protocol for mouse and human tissues (PMC7501639).
| Sample | Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet A13164-2) |
| Fixation | Image fixative and duration unreported (datasheet A13164-2); 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 A13164-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A13164-2) |
| Primary antibody | Rabbit anti-SYNE3, 2-5 μg/ml (datasheet A13164-2) |
| Primary incubation | Overnight at 4 °C (datasheet A13164-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A13164-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | SYNE3-positive staining in spermatogonia cells of testis (HPA tissue IHC: High). HPA tissue profile: Nuclear expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval (datasheet A13164-2). Use Tris/EDTA pH 9.0 when reproducing the published protocol (PMC7501639).
Section 2
What Is the Expected SYNE3 Staining Pattern?
SYNE3 is an outer nuclear membrane protein with a cytoplasmic region spanning residues 1–925 (UniProt Q6ZMZ3 topology). In paraffin-section IHC, expect nuclear-associated staining, especially in spermatogonia, where HPA reports High staining (HPA: Testis, spermatogonia High). HPA describes nuclear expression in several tissues, but rates its tissue IHC profile Approved with low consistency between antibody staining and RNA expression (HPA: tissue IHC reliability). Interpret additional positive cell populations cautiously.
| Strong staining outlines nuclei in spermatogonia within an otherwise interpretable testis section. | This fits the reported High spermatogonial staining (HPA: Testis, spermatogonia High) and nuclear membrane location (UniProt Q6ZMZ3; HPA: ICC-IF supported nuclear membrane). On chromogenic sections, assess nuclear association across multiple cells; a thin rim may be hard to resolve by light microscopy (general IHC practice). |
| Signal is predominantly diffuse cytoplasmic or uniformly fills nuclei, with no nuclear-associated pattern. | That distribution warrants caution because SYNE3 is assigned to the outer nuclear membrane and rough endoplasmic reticulum (UniProt Q6ZMZ3 subcellular location), while HPA supports a nuclear membrane location in ICC-IF (HPA: subcellular). Compare with controls and tissue structure before calling the stain specific (general IHC practice). |
| Strong staining appears in adipocytes or bone marrow hematopoietic cells. | HPA reports SYNE3 as Not detected in those cell populations (HPA: adipose adipocytes; bone marrow hematopoietic cells). Consider nonspecific antibody binding or endogenous chromogenic activity, then check the negative control and blocking steps (general IHC practice). A single unexpected positive result cannot establish expression. |
| Color covers broad tissue areas or appears equally in unrelated cell populations. | This is difficult to reconcile with a cell-associated, nuclear pattern (HPA: tissue IHC profile; UniProt Q6ZMZ3 location). Diffuse background can arise from antibody concentration, inadequate washing, or detection chemistry (general IHC practice). Judge specific staining only after the background is controlled. |
| Spermatogonia show no detectable signal, while tissue morphology and counterstain are preserved. | Treat this as an assay problem to investigate because HPA reports High staining in spermatogonia (HPA: Testis, spermatogonia High). Check the antibody's IHC validation, retrieval and detection setup, and a concurrently processed positive control (general IHC practice). HPA's low RNA-staining consistency limits broader expression inferences (HPA: reliability). |
💡Expected SYNE3 appearanceA convincing positive is High, nuclear-associated staining in testis spermatogonia (HPA: Testis, spermatogonia High; UniProt Q6ZMZ3 location); widespread diffuse color or strong staining in HPA Not detected cell populations calls for control review (HPA: tissue IHC; general IHC practice).
| Membrane topology | Residues 1–925 are cytoplasmic, 926–946 span the membrane, and 947–975 face the perinuclear space (UniProt Q6ZMZ3 topology). The antibody epitope is unspecified here, so topology alone cannot predict retrieval needs or staining intensity. |
| Tissue and cell selection | Testis spermatogonia provide the strongest listed IHC example (HPA: High). HPA reports several Not detected populations, including adipocytes and bone marrow hematopoietic cells (HPA: tissue IHC). Score the specified cell population rather than treating an entire tissue as uniformly positive or negative. |
| Antibody validation and expression agreement | HPA055227 is IHC Approved, whereas HPA077140 is ICC Supported (HPA: antibodies). The tissue IHC profile has low consistency with RNA expression (HPA: reliability). Neither status establishes that every antibody or every stained cell population has the same specificity. |
| Alternative isoforms | UniProt lists 3 SYNE3 isoforms (UniProt Q6ZMZ3 isoforms). No isoform-specific epitope or tissue distribution is supplied, so the record does not support assigning a staining difference to one isoform. |
| Retrieval and detection | Antigen retrieval and endogenous enzyme blocking are routine variables in chromogenic paraffin IHC (general IHC practice). No SYNE3-specific fixation sensitivity or retrieval condition is supplied; use the antibody's validated IHC instructions and matched controls without claiming a target-specific effect. |
| Situation | Likely cause | Next action |
|---|---|---|
| No staining in testis spermatogonia. | A failed IHC step or unsuitable antibody setup is possible; HPA reports High staining in these cells (HPA: Testis, spermatogonia High). | Check the IHC-validated antibody instructions, retrieval, dilution, detection reagents, and concurrent positive control (general IHC practice). Avoid inferring absence of SYNE3 from one failed section. |
| Strong signal in adipocytes or bone marrow hematopoietic cells. | These populations are Not detected in HPA tissue IHC (HPA: adipose tissue; bone marrow); nonspecific binding or endogenous detection activity is possible (general IHC practice). | Review a negative control, detection blocking, and cell identification; repeat under adjusted assay conditions if the unexpected signal persists (general IHC practice). |
| Color spreads across the section with little cell-level structure. | High antibody or detection background can obscure the nuclear-associated pattern (general IHC practice; HPA: tissue IHC profile). | Inspect controls, antibody dilution, wash steps, and chromogen development; score only signal distinguishable from background (general IHC practice). |
| Staining is mainly cytoplasmic or fills nuclei uniformly. | The pattern does not clearly match the outer nuclear membrane assignment (UniProt Q6ZMZ3 location) or supported nuclear membrane ICC-IF location (HPA: subcellular). | Recheck morphology, controls, and antibody validation before interpreting it as SYNE3; an apparent rim may be difficult to resolve in chromogenic sections (general IHC practice). |
| IHC looks positive in a cell population whose RNA result seems low. | HPA explicitly reports low consistency between antibody staining and RNA expression (HPA: tissue IHC reliability). | Report the observed IHC pattern with its cell identity and control results; do not use RNA agreement alone to validate or reject the stain (HPA: reliability; general IHC practice). |
| Does IF/ICC establish the expected compartment for this IHC result? | HPA supports nuclear membrane localisation in ICC-IF and lists REH, Rh30, and U2OS images (HPA: subcellular); its ICC Supported antibody differs from the IHC Approved antibody (HPA: antibodies). | Use IF/ICC as compartment context only: compare nuclear membrane localisation, while judging paraffin IHC with its own antibody validation and controls (HPA: subcellular; HPA: antibodies; general IHC practice). |
Experiment evidence
IHC & IF images showing SYNE3 staining
A13164-2 · IHCIHC analysis of Nesprin3/SYNE3 using anti-Nesprin3/SYNE3 antibody (A13164-2). Nesprin3/SYNE3 was detected in a paraffin-embedded section of
A13164-2 · IHCIHC analysis of Nesprin3/SYNE3 using anti-Nesprin3/SYNE3 antibody (A13164-2). Nesprin3/SYNE3 was detected in a paraffin-embedded section of
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000176438-SYNE3/tissue/cerebral+cortex#imid_15691671
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000176438-SYNE3/tissue/colon#imid_15691613
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000176438-SYNE3/tissue/liver#imid_15691614
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000176438-SYNE3/tissue/kidney#imid_15691599
HPA · ICC-IF REHHuman Protein Atlas immunofluorescence, REH, antibody HPA077140; green = SYNE3, blue = nucleus, red = microtubules.
HPA · ICC-IF REHHuman Protein Atlas immunofluorescence, REH, antibody HPA077140; green = SYNE3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for SYNE3 IHC & IF
🧪Run testis first and score spermatogonia for SYNE3 staining (HPA: High in spermatogonia). Use adipose tissue adipocytes as the negative comparison (HPA: Not detected in adipocytes); on the testis slide, treat neighboring cells lacking a distinct nuclear-rim signal as an internal background reference, without assuming they are SYNE3-negative (HPA: spermatogonia are the reported positive cells; HPA subcellular: nuclear membrane).
Positive control tissue: Testis (Spermatogonia cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show SYNE3 in REH, Rh30, U2OS, with annotated localisation: Nuclear membrane (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a rabbit isotype control matched to the primary antibody’s immunoglobulin class and clonality where applicable, and a validated SYNE3 knockout or peptide-block control (selected-SKU caption: rabbit primary; standard IHC controls). Quench endogenous peroxidase before chromogenic detection and check testis sections for background signal (selected-SKU caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU caption: fixative unreported). The caption uses heat-mediated retrieval in EDTA at pH 8.0, but does not establish that retrieval is required (selected-SKU caption: EDTA retrieval); IF may make the expected nuclear-rim pattern easier to assess, while an advantage for frozen sections and a testis-specific artefact are unreported (HPA subcellular: supported nuclear membrane localization; HPA: High in testis spermatogonia).
HPA tissue IHC evidence for SYNE3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low 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 |
|---|---|---|---|---|
| Testis | Spermatogonia cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced SYNE3 IHC Tips
Troubleshoot SYNE3 staining by checking retrieval, nuclear envelope localisation, epitope access and cell-specific controls before interpreting chromogenic signal (UniProt Q6ZMZ3; HPA tissue IHC).
Which retrieval condition should I try first for SYNE3 in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A13164-2). The selected image used this condition before overnight incubation at 4°C with 2 μg/ml primary antibody, so keep those conditions together for an initial comparison (datasheet A13164-2). If nuclear-envelope staining is weak, adjust heating duration on adjacent sections while holding antibody concentration and DAB development constant (standard IHC practice; HPA subcellular). Excess retrieval can damage tissue morphology and make a nuclear rim harder to distinguish, so compare signal with section integrity and a no-primary control (standard IHC practice; HPA subcellular).
How should I troubleshoot variable staining across differently fixed specimens?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative, and the supplied evidence provides no SYNE3 fixation comparison (datasheet A13164-2). Record each specimen's fixative and fixation duration, then compare adjacent sections processed with the same pH 8.0 EDTA retrieval and 2 μg/ml primary concentration (standard IHC practice; datasheet A13164-2). Include a consistently processed reference section in each staining run to separate specimen handling from assay variation (standard IHC practice). Assess preservation of cell and nuclear boundaries alongside staining intensity; damaged morphology makes the expected nuclear-envelope pattern difficult to score (HPA subcellular; standard IHC practice).
What staining pattern should count as plausible SYNE3 localisation?
Look for signal at the nuclear envelope, ideally a rim around nuclei, because SYNE3 localises to the outer nuclear membrane and HPA supports a nuclear-membrane location (UniProt Q6ZMZ3; HPA subcellular). Its cytoplasmic region spans residues 1–925, followed by a transmembrane segment at 926–946 and a perinuclear segment at 947–975 (UniProt Q6ZMZ3 topology). HPA describes nuclear expression in several tissues, but chromogenic resolution may not distinguish a thin envelope rim from adjacent nuclear staining (HPA tissue IHC; standard IHC practice). Compare well-preserved cells with a counterstain and avoid scoring diffuse DAB as specific solely because it overlaps nuclei (standard IHC practice).
Could isoform or epitope differences explain missing nuclear-rim staining?
SYNE3 has 3 reported isoforms, but the supplied antibody caption does not identify its epitope or establish which isoforms it detects (UniProt Q6ZMZ3; datasheet A13164-2). Interpret a negative section cautiously when the epitope's position and accessibility after heat retrieval are unknown (standard IHC practice; datasheet A13164-2). The annotated KASH domain occupies residues 917–975, crossing the 926–946 transmembrane segment, whereas residues 1–925 are cytoplasmic (UniProt Q6ZMZ3 topology). Check epitope information for the exact antibody and compare matched retrieval conditions before attributing a missing rim to isoform expression (standard IHC practice; UniProt Q6ZMZ3).
How can IF clarify an ambiguous chromogenic SYNE3 pattern?
Use IF as a separate localisation check: HPA supports a nuclear-membrane pattern and lists ICC/IF images from REH, Rh30 and U2OS cells (HPA subcellular). Multiplex SYNE3 with a marker identifying the expected cell type in the specimen, and choose a fluorophore channel with low tissue autofluorescence after inspecting unstained tissue (standard IF practice). Match permeabilisation to the antibody epitope: residues 1–925 face the cytoplasm, while 947–975 face the perinuclear space; the selected antibody's epitope is unspecified (UniProt Q6ZMZ3 topology; datasheet A13164-2). Compare nuclear-rim signal with single-channel and no-primary controls before using IF to interpret DAB staining (standard IF practice; HPA subcellular).
What should I check when DAB obscures the expected nuclear rim?
First compare a no-primary section and check endogenous peroxidase blocking, since enzyme activity can produce DAB signal independently of antibody binding (standard IHC practice). The selected paraffin-section image used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and peroxidase-linked detection with DAB (datasheet A13164-2). If diffuse staining persists, titrate primary concentration or shorten DAB development on adjacent sections while keeping retrieval at EDTA pH 8.0 (standard IHC practice; datasheet A13164-2). Judge improvements by whether nuclear-envelope signal remains distinct from cytoplasmic haze and tissue-edge deposits (HPA subcellular; standard IHC practice).
How should I score heterogeneous SYNE3 staining in paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored compartment as nuclear-envelope-associated staining before counting cells, using HPA's supported nuclear-membrane location as the localisation reference (HPA subcellular). Report the percentage of positive cells and an intensity-weighted H-score within each annotated cell population; use positive-cell density per mm² when cell abundance itself is relevant (standard IHC practice). Normalise counts to the number of eligible, intact cells or the assessed tissue area, and keep DAB exposure and scoring thresholds consistent across slides (standard IHC practice). Record cell type because HPA reports high staining in testis spermatogonia but no detection in several listed populations, and notes low staining–RNA consistency (HPA tissue IHC).
How can I distinguish genuine SYNE3 signal from staining artefact?
Prioritise a nuclear-envelope pattern in intact cells, consistent with SYNE3's outer-nuclear-membrane localisation and HPA's supported nuclear-membrane assignment (UniProt Q6ZMZ3; HPA subcellular). Compare the stained cell population with the tissue context: HPA reports high signal in testis spermatogonia and no detection in adipocytes in adipose tissue, among other listed populations (HPA tissue IHC). Exclude section edges, necrotic regions and DAB signal reproduced in a no-primary or inadequately peroxidase-blocked control (standard IHC practice). Treat unexpected staining cautiously because HPA rates the tissue assay Approved while reporting low consistency between antibody staining and RNA expression (HPA tissue IHC).
Boster reagents
Best SYNE3 / Nesprin-3 IHC Antibodies
The IHC-validated anti-SYNE3 antibody has real paraffin-section images from human liver cancer, thyroid papillary carcinoma and spleen rupture tissue, and mouse liver (A13164-2 image captions).
Real IHC data
Anti-Nesprin3/SYNE3 Antibody ®
Cat # A13164-2
A13164-2 is listed for IHC in human and mouse (catalog: applications and reactivity). Its IHC images show paraffin sections of human liver cancer, thyroid papillary carcinoma and spleen rupture tissue, and mouse liver (A13164-2 image captions).
Which to pick: Choose A13164-2 for paraffin-section IHC in human tissue or mouse liver; its captions document EDTA retrieval at pH 8.0 and primary antibody at 2 μg/ml (A13164-2 image captions). The fixative is unreported (A13164-2 image captions). No IF/ICC-validated SKU or IF image is supplied, so an IF/ICC choice would require separate validation; A13164-2 is the listed cross-species IHC option for human and mouse (catalog: applications, reactivity and IF images).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q6ZMZ3 (SYNE3_HUMAN, Nesprin-3).
- Human Protein Atlas. SYNE3 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. SYNE3 subcellular location (ICC-IF): Localized to the nuclear membrane..
- Human Protein Atlas. SYNE3 antibody validation summary (2 antibodies).
- Expression profile of SYNE3 and bioinformatic analysis of its prognostic value and functions in tumors. Journal of translational medicine 2020 — PMC7501639.
- PubMed PMID:12508121 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for SYNE3 (Nesprin-3). Expected localisation Nuclear staining in tissue (HPA tissue IHC); outer nuclear membrane location (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A13164-2). Validated IHC antibody: A13164-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Spermatogonia cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet A13164-2); 2. Fixation: Image fixative and duration unreported (datasheet A13164-2); 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 A13164-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A13164-2); 8. Primary antibody: Rabbit anti-SYNE3, 2-5 μg/ml (datasheet A13164-2); 9. Primary incubation: Overnight at 4 °C (datasheet A13164-2). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A13164-2); Caveat: Bone marrow RNA is enhanced, but hematopoietic IHC is undetected (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope differences unspecified (UniProt); Regulation: Tissue-enhanced RNA in bone marrow (HPA tissue RNA). Evidence: UniProt Q6ZMZ3, HPA tissue IHC · Approved, HPA ICC-IF · nuclear membrane., Datasheet A13164-2, PMC7501639. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/SYNE3/immunohistochemistry-ihc-guide.