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CERS6 / Ceramide synthase 6 · IHC design guide
Design Immunohistochemistry for CERS6
Plan CERS6 paraffin IHC using cerebellar granular layer cells, which show high staining (HPA tissue IHC), and the catalog antibody starting at 2.5 μg/mL (datasheet). Interpret the reported nuclear tissue staining (HPA tissue IHC) alongside the ER membrane location (UniProt) and low staining–RNA consistency (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CERS6 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 tissue staining (HPA tissue IHC); ER membrane (UniProt) | |
| Staining pattern | Nuclear staining in several tissues (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Cerebellum+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Low consistency between staining and RNA expression (HPA tissue IHC) | |
| Regulation | Staining regulation uncharacterized (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage across ER faces is unknown (UniProt) |
Section 1
Recommended CERS6 IHC & IF Protocols
The catalog antibody protocol and one published CERS6 IHC protocol are available for paraffin sections (page retrieval specification; PMC4695011: methods).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A07033); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-CERS6, 2.5 μg/mL (datasheet A07033) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CERS6-positive staining in cells in granular layer of cerebellum (HPA tissue IHC: High). HPA tissue profile: Nuclear expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval for the catalog antibody (page retrieval specification); the published xenograft protocol used pH 8.4 retrieval (PMC4695011: methods).
Section 2
What Is the Expected CERS6 Staining Pattern?
CERS6 is a 7-pass endoplasmic reticulum membrane protein (UniProt Q6ZMG9 topology). In IHC, the strongest reported signal is in cerebellar granular-layer cells; several other cell populations show medium staining (HPA tissue IHC). HPA describes nuclear expression in several tissues, which conflicts with the UniProt location and remains unresolved (HPA tissue IHC; UniProt Q6ZMG9 location). HPA rates its IHC antibody Approved but reports low consistency with RNA expression (HPA tissue IHC).
| Granular-layer cells in cerebellum stain strongly, while nearby structures can be distinguished on the counterstained section (HPA: High in cerebellar granular-layer cells). | This is the clearest supplied tissue-level positive reference. Score signal within the named cells and record its compartment separately; HPA's reported nuclear pattern and UniProt's ER location do not establish one concordant subcellular readout (HPA tissue IHC; UniProt Q6ZMG9 location). |
| Hematopoietic cells in bone marrow, neuronal cells in cerebral cortex, or glandular cells in fallopian tube show moderate staining (HPA: Medium in each listed cell population). | These patterns are consistent with the reported IHC distribution. Compare like cell types rather than calling an entire section positive; medium HPA staining is an observation, not proof that each stained structure contains CERS6 (HPA tissue IHC: Approved; low RNA concordance). |
| A crisp plasma-membrane rim or exclusive mitochondrial pattern dominates the candidate positive cells (UniProt Q6ZMG9: ER membrane). | Treat the compartment mismatch as a possible artefact and investigate before scoring it as CERS6. HPA also reports nuclear IHC staining, so an exclusively nuclear pattern needs separate validation rather than automatic acceptance or rejection (HPA tissue IHC; UniProt Q6ZMG9 location). |
| Strong stain appears in adipocytes or cardiomyocytes, or colour fills many cell types uniformly (HPA: Not detected in adipocytes and cardiomyocytes). | This diverges from the supplied cell-type observations and raises cross-reactivity or detection-background concerns. HPA's Not detected calls apply to the specified cells and tested IHC conditions; they are not universal proof of biological absence (HPA tissue IHC). |
| A section has diffuse haze, or a cerebellar granular layer has no discernible stain (HPA: High in granular-layer cells). | Diffuse haze limits compartment and cell-type scoring; absence in the strongest reported reference makes the run hard to interpret. Use matched controls and inspect the staining workflow before inferring low CERS6 abundance (HPA tissue IHC; general IHC practice). |
💡Expected CERS6 appearanceA defensible positive is high staining in cerebellar granular-layer cells, with the compartment explicitly recorded; an ER-associated cellular pattern fits UniProt, while HPA-reported nuclear staining requires independent scrutiny. Uniform colour in HPA Not detected cell types is a warning sign (HPA tissue IHC; UniProt Q6ZMG9 location).
| Subcellular location and topology (UniProt Q6ZMG9) | CERS6 is an ER membrane protein with 7 transmembrane segments and lumenal and cytoplasmic regions. An antibody's epitope position, which is not supplied, matters when interpreting access after section preparation (UniProt Q6ZMG9 topology; general IHC practice). |
| Reported tissue distribution (HPA tissue IHC) | Cerebellar granular-layer cells are High; bone-marrow hematopoietic cells, cortical neurons, hippocampal glia, fallopian-tube glandular cells and smooth-muscle cells are Medium. Choose comparison fields by cell identity and reported level (HPA tissue IHC). |
| Observed nuclear IHC versus annotated ER location (HPA tissue IHC; UniProt Q6ZMG9) | HPA's profile says nuclear expression in several tissues, whereas UniProt assigns CERS6 to the ER membrane. Keep nuclear and cytoplasmic scores separate; these sources alone cannot resolve antibody specificity or true localisation (HPA tissue IHC; UniProt Q6ZMG9 location). |
| Strength of antibody evidence (HPA antibodies; HPA tissue IHC) | HPA044683 is listed as IHC Approved, with low consistency between staining and RNA data; no IHC Enhanced status is supplied. Treat tissue images as observational references, especially for unexpected compartments (HPA antibodies; HPA tissue IHC). |
| Molecular forms and site information (UniProt Q6ZMG9) | UniProt lists 2 isoforms, one N-linked glycosylation site at residue 18, and a full-length 1–384 chain. The supplied record does not identify the antibody epitope or show isoform-specific IHC staining (UniProt Q6ZMG9). |
| ICC/IF evidence boundary (HPA subcellular; HPA antibodies) | HPA supplies no main ICC/IF location, no imaged cell lines, and no ICC status for HPA044683. An IHC pattern therefore cannot serve as an established IF/ICC localisation standard (HPA subcellular; HPA antibodies). |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in cerebellar granular-layer cells (HPA: High). | The expected reference is absent; section quality, detection performance or antibody use may be responsible, but the supplied sources do not identify a CERS6-specific failure mechanism (HPA tissue IHC; general IHC practice). | Run a known-positive cerebellum section alongside a no-primary control, verify the chromogenic detection reagents, and review the antibody's IHC-P instructions before interpreting the specimen (HPA tissue IHC; general IHC practice). |
| Nuclear staining dominates without a clear ER-associated pattern (HPA: nuclear expression; UniProt Q6ZMG9: ER membrane). | This matches HPA's reported appearance but conflicts with the UniProt location; HPA also reports low staining-to-RNA consistency, so specificity remains uncertain (HPA tissue IHC; UniProt Q6ZMG9 location). | Record nuclear and cytoplasmic scores separately, compare HPA-reported positive and Not detected cell populations, and seek an independent specificity control before calling nuclear CERS6 (HPA tissue IHC; general IHC practice). |
| Adipocytes or cardiomyocytes stain strongly (HPA: Not detected in these cells). | The signal conflicts with HPA's cell-specific observations; cross-reactivity or endogenous chromogenic activity is possible, especially if colour also appears in control sections (HPA tissue IHC; general IHC practice). | Check the same cell types in a no-primary control, assess endogenous enzyme blocking where relevant, and compare signal with a reported positive tissue on the same run (HPA tissue IHC; general IHC practice). |
| Diffuse colour obscures cell borders and the compartment cannot be scored (general IHC practice). | Background can arise from nonspecific reagent binding or the detection system; a diffuse field is not evidence for CERS6 distribution (general IHC practice). | Inspect the no-primary control, review blocking and washing, and adjust the IHC-validated antibody only within its supplied instructions; rescore once individual cells can be resolved (general IHC practice). |
| A weak result appears in lung macrophages or thyroid glandular cells (HPA: Low in these cells). | Low reported staining can be difficult to distinguish from background, and HPA's low RNA concordance limits confidence in a faint isolated signal (HPA tissue IHC). | Compare with a strong cerebellar reference and the no-primary control from the same run; report equivocal faint colour as such instead of promoting it to a firm positive (HPA tissue IHC; general IHC practice). |
| Can this IHC pattern be used as an IF/ICC protocol or localisation benchmark? (HPA subcellular; HPA antibodies). | The supplied HPA subcellular record has no main IF location or imaged cell lines, and HPA044683 has no listed ICC status (HPA subcellular; HPA antibodies). | Use the separate IF/ICC guide for application-specific design and validation; treat ER localisation as a UniProt-based expectation and nuclear IHC as an unresolved HPA observation (UniProt Q6ZMG9 location; HPA tissue IHC). |
Experiment evidence
IHC & IF images showing CERS6 staining
A07033 · IHCImmunohistochemistry of LASS6 in mouse brain tissue with LASS6 antibody at 2.5 μg/mL.
A07033 · IFImmunofluorescence of LASS6 in Mouse Brain cells with LASS6 antibody at 5 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000172292-CERS6/tissue/cerebral+cortex#imid_12083179
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000172292-CERS6/tissue/colon#imid_12083113
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000172292-CERS6/tissue/liver#imid_12083114
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000172292-CERS6/tissue/kidney#imid_12083115
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CERS6 IHC & IF
🧪Run cerebellum first and score cells in the granular layer, where CERS6 staining is High (HPA: cerebellum, cells in granular layer). Run heart muscle as a negative comparator because cardiomyocytes are Not detected (HPA: heart muscle); on the positive slide, use cells without specific chromogen as internal background comparators without assuming their identity establishes CERS6 negativity.
Positive control tissue: Cerebellum (Cells in granular layer, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for CERS6; derive a cell-line control from the positive tissue's cell type (Cells in granular layer) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control and an irrelevant antibody matched to the primary antibody’s host and immunoglobulin class, with clonality matched where applicable (standard IHC practice). Use CERS6 knockout material as a biological negative, and block endogenous peroxidase before chromogenic detection (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence; the A07033 mouse-brain IHC caption gives 2.5 μg/mL but does not report the fixative (selected tissue-IHC caption). Retrieval dependence is unreported, so compare retrieval conditions on pilot paraffin sections (standard IHC practice). The supplied evidence does not establish that frozen sections or IF are easier; dense granular-layer cellularity may complicate cell-by-cell scoring (HPA: High in cerebellar granular-layer cells).
HPA tissue IHC evidence for CERS6
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 |
|---|---|---|---|---|
| Cerebellum | Cells in granular layer | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | Medium | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | Medium | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CERS6 IHC Tips
Troubleshoot CERS6 staining in paraffin sections by checking retrieval, antibody specificity, cellular compartment and controls before interpreting chromogenic signal.
What retrieval should I start with for CERS6 in paraffin sections?
Use citrate buffer at pH 6.0 for HIER at 95–98 °C for 20 min (page retrieval setting). Keep section thickness, cooling time and detection conditions constant while comparing retrieval runs, so a change in signal has a clear cause (standard IHC practice). CERS6 has 7 transmembrane segments and resides in the endoplasmic reticulum membrane, making epitope accessibility a practical concern (UniProt Q6ZMG9 topology and subcellular location). If staining remains weak, test a different retrieval condition on matched sections only after confirming that the catalog antibody works with the specified citrate method (standard IHC practice).
How should I assess whether fixation is reducing CERS6 staining?
Target-specific fixation sensitivity is unknown because the supplied CERS6 evidence does not specify a fixative or a fixation comparison (selected A07033 caption; supplied evidence). The mouse brain image reports antibody staining at 2.5 µg/mL, but its caption leaves the fixative unstated (A07033 tissue-IHC caption). For a controlled assessment, process matched samples in 10% neutral buffered formalin for defined, recorded intervals, then hold retrieval and detection constant across sections (standard IHC practice). Compare signal in the same cell population and examine morphology alongside staining; differences between fixation conditions establish a local assay effect, not a general CERS6 fixation requirement (standard IHC practice).
What cellular staining pattern is plausible for CERS6?
CERS6 is an endoplasmic reticulum membrane protein with 7 predicted transmembrane segments, so a cellular pattern compatible with intracellular membranes is plausible (UniProt Q6ZMG9 subcellular location and topology). Its annotated cytoplasmic and lumenal stretches mean the accessible side depends on the antibody epitope, which is not specified here (UniProt Q6ZMG9 topology; supplied evidence). The tissue atlas instead describes nuclear staining in several tissues, while warning of low consistency between staining and RNA expression (HPA tissue IHC). Treat predominantly nuclear chromogen as a finding needing independent specificity checks rather than assigning it to the annotated ER location (UniProt Q6ZMG9 subcellular location; HPA tissue IHC; standard IHC practice).
How can epitope placement affect CERS6 IHC interpretation?
The record lists 2 CERS6 isoforms, but no antibody epitope or isoform coverage is supplied, so staining cannot be assigned to one isoform from this evidence (UniProt Q6ZMG9 isoforms; supplied evidence). CERS6 spans the membrane 7 times, with alternating lumenal and cytoplasmic regions that could differ in accessibility after processing (UniProt Q6ZMG9 topology). A glycosylation site is annotated at residue 18; its relevance to staining depends on whether the unknown epitope overlaps that region (UniProt Q6ZMG9 glycosylation; supplied evidence). Check the antibody's documented immunogen or epitope and compare with an independent specificity control before interpreting regional staining differences as isoform expression (standard IHC practice).
How should I check CERS6 by multiplex IF alongside this IHC assay?
For a separate IF assessment, pair CERS6 with a marker identifying the cell population being examined, such as a neuronal marker when evaluating cerebral cortex neuronal cells (HPA: Medium in cerebral cortex neuronal cells; standard IF practice). Select spectrally separated fluorophores and place a weak target signal in a channel with low measured tissue autofluorescence; include unstained tissue to inspect that background (standard IF practice). CERS6 has both cytoplasmic and lumenal regions across 7 transmembrane segments, while the antibody epitope is unspecified (UniProt Q6ZMG9 topology; supplied evidence). Optimise permeabilisation against epitope access and preservation of ER structure, then compare compartment patterns with the chromogenic result (standard IF practice; UniProt Q6ZMG9 subcellular location).
How can I distinguish CERS6 chromogen from background?
Run a no-primary control through the same secondary detection and DAB development, and block endogenous peroxidase before detection (standard chromogenic IHC practice). Inspect pigment, tissue folds, section edges and damaged regions at the same exposure or microscope settings used for scored fields (standard IHC practice). The selected mouse brain image documents staining at 2.5 µg/mL, but does not establish that this concentration minimises background in other preparations (A07033 tissue-IHC caption). Titrate the catalog antibody on matched sections around a documented working concentration, keeping citrate pH 6.0 retrieval and development time fixed while assessing signal over the no-primary control (page retrieval setting; standard IHC practice).
How should I quantify CERS6 staining across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because CERS6 is annotated at the ER membrane while tissue-atlas IHC reports nuclear expression (UniProt Q6ZMG9 subcellular location; HPA tissue IHC). For comparable regions, record either an H-score from intensity and percentage of positive cells, or positive cells per mm² when cell density is the question (standard IHC practice). Normalise cell counts to viable tissue area or total eligible cells of the same type, and use identical thresholds and DAB development across samples (standard IHC practice). Report nuclear and extranuclear signal separately; combining them would obscure the compartment discrepancy that needs validation (UniProt Q6ZMG9 subcellular location; HPA tissue IHC; standard IHC practice).
When should I doubt an apparent CERS6-positive IHC result?
Question a predominantly nuclear result because CERS6 is annotated as an ER membrane protein, although the tissue atlas reports nuclear staining and flags low staining–RNA consistency (UniProt Q6ZMG9 subcellular location; HPA tissue IHC). Compare the same cell type across sections: the atlas reports high staining in cerebellar granular-layer cells and no detected staining in adipocytes of adipose tissue (HPA tissue IHC). Exclude edge-enhanced signal, necrotic areas and staining retained in a no-primary control before calling cells positive (standard IHC practice). Check endogenous peroxidase contribution with an appropriate block and require reproducible cellular staining before drawing a biological conclusion from DAB intensity (standard chromogenic IHC practice).
Boster reagents
Best CERS6 / Ceramide synthase 6 IHC Antibodies
A07033 has IHC data from mouse brain tissue and IF data from mouse brain cells (A07033 image captions). The catalog lists Human, Mouse, and Rat reactivity (A07033 catalog reactivity).
Real IHC data
Anti-LASS6 CERS6 Antibody
Cat # A07033
A07033 will render with its mouse brain tissue IHC figure at 2.5 μg/mL (A07033 IHC image caption). Its separate IF caption shows mouse brain cells at 5 μg/mL; the catalog lists IHC-P and IF applications (A07033 IF image caption; catalog applications).
Which to pick: For tissue IHC, choose A07033: its own caption shows mouse brain tissue staining, and the catalog lists IHC-P; the fixative is unreported (A07033 IHC image caption; catalog applications). For IF/ICC, A07033 is the IF-listed option with a mouse brain cell image (A07033 catalog applications; IF image caption). For work across species, the catalog lists Human, Mouse, and Rat reactivity for A07033; its host is Rabbit and its clonality is unreported (A07033 catalog reactivity, host, and clone fields).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q6ZMG9 (CERS6_HUMAN, Ceramide synthase 6).
- Human Protein Atlas. CERS6 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. CERS6 subcellular location (ICC-IF): Variation in transcript expression is not correlated to the cell cycle..
- Human Protein Atlas. CERS6 antibody validation summary (1 antibodies).
- CERS6 promotes esophageal squamous cell carcinoma proliferation by increasing the stability of RPN1. Cell death discovery 2025 — PMC12594893.
- CERS6 required for cell migration and metastasis in lung cancer. Journal of cellular and molecular medicine 2020 — PMC7579715.
- TGF-β receptor I/II trafficking and signaling at primary cilia are inhibited by ceramide to attenuate cell migration and tumor metastasis. Science signaling 2017 — PMC5818989.
- By activating Fas/ceramide synthase 6/p38 kinase in lipid rafts, stichoposide D inhibits growth of leukemia xenografts. Oncotarget 2015 — PMC4695011.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15815621 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CERS6 (Ceramide synthase 6). Expected localisation Nuclear tissue staining (HPA tissue IHC); ER membrane (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A07033 at 2.5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Cerebellum (Cells in granular layer); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet A07033); 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-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-CERS6, 2.5 μg/mL (datasheet A07033); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Low consistency between staining and RNA expression (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage across ER faces is unknown (UniProt); Regulation: Staining regulation uncharacterized (UniProt). Evidence: UniProt Q6ZMG9, HPA tissue IHC · Approved, HPA cell-line RNA · Variation in transcript expression is not correlated to the cell…, Datasheet A07033, PMC4695011. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CERS6/immunohistochemistry-ihc-guide.