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CERS5 / Ceramide synthase 5 · IHC design guide
Design Immunohistochemistry for CERS5
Plan CERS5 paraffin IHC using the IHC-validated A08612-1 antibody at a starting concentration of 2.5 μg/mL (datasheet). Compare high staining in testis Sertoli cells with undetected staining in heart cardiomyocytes, and assess the observed cytoplasmic and nucleolar pattern alongside the ER membrane annotation (HPA tissue IHC; UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CERS5 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 | ER membrane (UniProt); cytoplasmic/nucleolar tissue staining (HPA tissue IHC) | |
| Staining pattern | Sertoli cells: high; cytoplasmic/nucleolar tissue pattern (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Testis+4 more · see all | |
| Negative control | Heart muscle+1 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); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A08612-1) | |
| Caveat | Nucleolar staining warrants validation against the ER expectation (HPA tissue IHC; UniProt) | |
| Regulation | PAQR4 regulates CERS5 stability (UniProt) | |
| Isoform / epitope | 2 isoforms; check luminal versus cytoplasmic epitope coverage (UniProt) |
Section 1
Recommended CERS5 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with published gastric and colorectal tissue protocols (PMC9552061; PMC4858121).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A08612-1); 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-CERS5, 2.5 μg/mL (datasheet A08612-1) |
| 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 | CERS5-positive staining in sertoli cells of testis (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and nucleolar expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval rule); the colorectal protocol also used citrate for 20 min (PMC4858121).
Section 2
What Is the Expected CERS5 Staining Pattern?
CERS5 is an endoplasmic reticulum membrane protein with 6 transmembrane segments (UniProt Q8N5B7 topology). In tissue IHC, expect cytoplasmic staining, sometimes with nucleolar staining, across several tissues (HPA: tissue IHC profile). Sertoli cells show the strongest listed signal; several glandular and glial cell populations show medium signal (HPA: High in Sertoli cells; Medium in listed glandular and glial cells). HPA rates its tissue IHC profile Approved, with medium consistency between staining and RNA expression (HPA: reliability).
| Strong cytoplasmic signal in testis Sertoli cells, with possible nucleolar staining. | This matches the strongest listed tissue result and the reported tissue IHC compartments (HPA: High in Sertoli cells; cytoplasmic and nucleolar profile). Score the cells and compartments separately: a strong Sertoli cell result does not mean every cell in the section should stain equally (HPA: cell-specific tissue IHC data). |
| Moderate staining in glandular cells or cerebral cortex glial cells. | This is compatible with the listed medium signals in adrenal gland, appendix, breast, colon and duodenum glandular cells, and cerebral cortex glial cells (HPA: Medium in these cell populations). Compare like cell types when scoring; a field-wide intensity score can obscure the reported cell-specific pattern (general IHC practice). |
| Uniform nuclear staining without a discernible cytoplasmic or nucleolar pattern. | Treat this as questionable compartment assignment rather than confirmation of CERS5: tissue IHC reports cytoplasmic and nucleolar expression, while UniProt places CERS5 at the endoplasmic reticulum membrane (HPA: tissue IHC profile; UniProt Q8N5B7 subcellular location). Review morphology and controls before calling it an artefact; nucleolar signal itself is reported by HPA (HPA: tissue IHC profile). |
| Strong staining in cardiomyocytes or smooth muscle cells. | These cell types are listed as not detected in the sampled tissue IHC data (HPA: heart muscle cardiomyocytes; smooth muscle cells). Unexpected strong signal raises concern for cross-reactivity or endogenous detection activity (general IHC practice). Recheck controls and staining distribution; an HPA non-detection is a comparison point, not proof that all specimens must be negative (HPA: tissue IHC data). |
| Hazy, widespread color across cells and tissue spaces. | Diffuse background makes compartment and cell-type calls unreliable (general IHC practice). It does not reproduce the reported cytoplasmic and nucleolar, cell-dependent profile (HPA: tissue IHC profile). Inspect the negative detection control and tissue morphology before interpreting weak color as CERS5 (general IHC practice). |
💡Expected CERS5 appearanceCall a result consistent when Sertoli cells show high cytoplasmic staining, with possible nucleolar signal (HPA: High in Sertoli cells; tissue IHC profile); uniform dark staining across unrelated cells or compartments is suspect (general IHC practice).
| Which compartment should guide IHC scoring? | Use the observed cytoplasmic and nucleolar tissue pattern for IHC interpretation (HPA: tissue IHC profile). The endoplasmic reticulum membrane assignment and 6 transmembrane segments provide molecular context, but do not establish how every chromogenic section will resolve the protein (UniProt Q8N5B7 topology; general IHC practice). |
| How much should intensity vary by cell type? | The supplied IHC observations range from High in Sertoli cells to Medium in several glandular or glial populations, Low in listed respiratory, neural and lung populations, and Not detected in cardiomyocytes and smooth muscle cells (HPA: tissue IHC data). Choose a control and scoring threshold with its specific cell population in mind (general IHC practice). |
| What does antibody validation support? | The tissue IHC profile is Approved, with medium consistency between antibody staining and RNA expression (HPA: reliability). HPA026589 is IHC Approved, whereas HPA006780 has an ICC approval listed and no IHC status in the supplied antibody record (HPA: antibody validation). Do not transfer an ICC approval to IHC (HPA: antibody validation). |
| IF/ICC: should its location be read as the IHC pattern? | HPA ICC-IF reports mainly nuclear membrane localization, with additional endoplasmic reticulum and primary cilium localization (HPA: subcellular ICC-IF). Tissue IHC separately reports cytoplasmic and nucleolar staining (HPA: tissue IHC profile). Interpret each assay against its own reported pattern; this section does not establish an IF/ICC staining protocol (HPA: assay-specific observations). |
| Do topology or isoforms predict epitope access? | UniProt lists 2 isoforms, a lumenal N-terminus and a cytoplasmic C-terminus (UniProt Q8N5B7 isoforms; topology). The supplied record gives no antibody epitope, so it cannot predict which isoform is recognized or whether a particular retrieval condition improves staining (UniProt Q8N5B7 record; general IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in testis Sertoli cells. | A known high-staining comparison population is missing (HPA: High in Sertoli cells); a detection or tissue-processing problem is possible (general IHC practice). | Check that Sertoli cells are present and assess a matched positive control, primary-antibody incubation, retrieval condition and detection reagents (general IHC practice). Do not infer a CERS5-specific fixation sensitivity from this result (HPA: tissue IHC scope). |
| Only faint staining in a chosen control. | The chosen cells may have Low rather than High reported staining; lung macrophages and bronchial respiratory epithelial cells are listed Low (HPA: tissue IHC data). | Compare the same identified cell type across sections, or include the listed High Sertoli cell population to check assay performance (HPA: tissue IHC data; general IHC practice). Avoid treating every faint tissue as a failed run. |
| Color persists without primary antibody. | Signal can arise from the chromogenic detection workflow or endogenous activity (general IHC practice). | Review the negative detection control; apply appropriate endogenous-enzyme blocking and check detection reagents according to the chromogen system (general IHC practice). Reassess CERS5 only after background is controlled. |
| Strong signal appears in cardiomyocytes or smooth muscle cells. | This conflicts with the listed Not detected observations and may reflect nonspecific binding or detection background (HPA: heart muscle and smooth muscle IHC; general IHC practice). | Compare a primary-omission control, inspect the exact stained cell type and optimize blocking or antibody concentration as general IHC adjustments (general IHC practice). Treat the HPA observations as reference data, not absolute exclusions. |
| Nuclear staining dominates the section. | Nucleolar staining is reported, but uniform nuclear filling does not match the stated cytoplasmic and nucleolar tissue profile (HPA: tissue IHC profile). | Use morphology and counterstain to distinguish nucleoli from whole nuclei; compare a positive tissue and negative detection control before assigning specificity (general IHC practice). Keep the ICC-IF nuclear membrane result distinct from tissue IHC (HPA: subcellular ICC-IF). |
| Results differ after changing antigen retrieval. | Retrieval can alter general IHC signal and background, but target-specific retrieval or fixation sensitivity is unreported in the supplied CERS5 evidence (general IHC practice; UniProt Q8N5B7 and HPA scope). | Compare retrieval conditions on matched sections with the same positive and negative controls, then score the expected cells and compartments (general IHC practice; HPA: tissue IHC profile). Avoid claiming that one condition reveals a proven CERS5 epitope. |
Experiment evidence
IHC & IF images showing CERS5 staining
A08612-1 · IHCImmunohistochemistry of LASS5 in human brain tissue with LASS5 antibody at 2.5 μg/mL.
A08612-1 · IFImmunofluorescence of LASS5 in Human Brain cells with LASS5 antibody at 20 μg/mL.
A08612 · IFImmunofluorescence of LASS5 in human brain tissue with LASS5 antibody at 20 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000139624-CERS5/tissue/cerebral+cortex#imid_6509816
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000139624-CERS5/tissue/colon#imid_6513190
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000139624-CERS5/tissue/liver#imid_6513207
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000139624-CERS5/tissue/kidney#imid_6513192
HPA · ICC-IF SK-MEL-30Human Protein Atlas immunofluorescence, SK-MEL-30, antibody HPA006780; green = CERS5, blue = nucleus, red = microtubules.
HPA · ICC-IF SK-MEL-30Human Protein Atlas immunofluorescence, SK-MEL-30, antibody HPA006780; green = CERS5, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CERS5 IHC & IF
🧪Run testis first and require staining in Sertoli cells (HPA: High in Sertoli cells). Use heart muscle as the negative tissue (HPA: Not detected in cardiomyocytes); on the testis slide, treat other cells as internal negative comparators only when their signal matches the no-primary background.
Positive control tissue: Testis (Sertoli cells, HPA High)
Negative control tissue: Heart muscle (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CERS5 in SK-MEL-30, U2OS, hTERT-RPE1, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), with annotated localisation: Nuclear membrane (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a species- and clonality-matched isotype control; confirm specificity with CERS5 knockout material or an immunizing-peptide block when available (standard IHC practice). For testis IHC, block endogenous peroxidase and check the no-primary slide for residual chromogen; if using IF, assess autofluorescence on an unstained section (standard IHC/IF practice).
⚠️Feasibility: A CERS5-specific fixation window and antigen-retrieval dependency are unreported in the supplied evidence; compare retrieval conditions during paraffin-section optimization (standard IHC practice). The selected A08612-1 human brain IHC caption reports 2.5 µg/mL but leaves the fixative unreported (catalog caption). HPA lists ICC-IF images and CERS5 localization at the nuclear membrane, endoplasmic reticulum, and primary cilium, but the supplied evidence does not establish that frozen sections or IF are easier than paraffin IHC (HPA: subcellular); assess testis background with the controls above (standard IHC/IF practice).
HPA tissue IHC evidence for CERS5
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 |
|---|---|---|---|---|
| Testis | Sertoli cells | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | Glial cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced CERS5 IHC Tips
Troubleshoot CERS5 staining in paraffin sections by checking retrieval, compartment pattern, cell identity and controls before comparing signal intensity.
How should I retrieve CERS5 antigen in paraffin sections?
Start with citrate buffer at pH 6.0 for heat induced retrieval at 95–98 °C for 20 min (page retrieval setting). Let sections cool in buffer, then wash before blocking; keep section thickness, heating vessel and cooling time consistent across comparison slides (standard IHC practice). If staining is weak, test a second retrieval condition on adjacent sections while holding antibody concentration and detection constant, and check whether tissue structure remains intact (standard IHC practice). The catalog brain image reports 2.5 µg/mL antibody but does not state its fixative or retrieval method, so it cannot establish retrieval performance for your sections (A08612-1 caption).
How can I distinguish a fixation problem from weak CERS5 expression?
Target specific fixation sensitivity is unknown from the supplied evidence; the catalog brain tissue caption does not state a fixative (A08612-1 caption). Record fixation duration, processing history and section age, then compare matched sections processed together with the same retrieval and antibody concentration (standard IHC practice). Include a reference tissue with an expected positive cell population, such as testis Sertoli cells, which show high staining, and inspect its cellular pattern alongside your sample (HPA: High in Sertoli cells). If both lose signal, investigate processing or detection before assigning a biological difference; if only one differs, repeat with independently processed material (standard IHC practice).
Which staining compartments are plausible for CERS5?
Assess a cytoplasmic or perinuclear membrane pattern first because CERS5 is annotated as an endoplasmic reticulum membrane protein with 6 transmembrane segments (UniProt Q8N5B7 topology and subcellular annotation). Tissue IHC also reports cytoplasmic and nucleolar staining, while subcellular imaging places CERS5 mainly at the nuclear membrane and additionally at the endoplasmic reticulum and primary cilium (HPA tissue IHC; HPA subcellular). These observations support examining compartment patterns across cells, but they do not make every isolated nuclear dot a confirmed CERS5 signal (HPA tissue IHC; standard IHC practice). Compare the pattern with a no primary control and with appropriately stained reference tissue before interpreting unusual compartments (standard IHC practice).
What should I check if an antibody misses one CERS5 form?
CERS5 has 2 annotated isoforms, but the supplied evidence does not map this antibody’s epitope onto either sequence (UniProt Q8N5B7 isoforms; A08612-1 caption). Request the immunogen or epitope information and check whether it falls in a sequence shared by both forms before claiming that IHC measures total CERS5 (standard antibody validation practice). Topology places residues 1–46 lumenally and 332–392 cytoplasmically, with 6 membrane spanning segments, so epitope position could affect access after processing (UniProt Q8N5B7 topology). If forms must be distinguished, use an independently validated form specific reagent or orthogonal assay and report which sequence it recognizes (standard antibody validation practice).
How should I assess CERS5 by IF alongside a cell identity marker?
Treat IF as a separately optimized application: the catalog brain image documents tissue IHC at 2.5 µg/mL, not an IF working concentration (A08612-1 caption). Multiplex CERS5 with a validated marker for the cell population under study; glial cells in cerebral cortex are one tissue reference for CERS5 staining (HPA: Medium in cerebral cortex glial cells). Choose spectrally separated fluorophores after imaging unstained tissue, placing the weaker channel where autofluorescence is lowest and checking single stain controls (standard IF practice). Match permeabilisation to the mapped epitope: the N terminus is lumenal and the C terminus cytoplasmic, but this antibody’s epitope is unreported (UniProt Q8N5B7 topology; A08612-1 caption).
How can I reduce diffuse or granular background in CERS5 IHC?
Run a no primary control and inspect unstained tissue before changing antibody concentration; these reveal detection background and native pigment that can resemble chromogen (standard IHC practice). For peroxidase detection, include a peroxidase block, then optimize protein blocking and washes while keeping retrieval identical across test sections (standard chromogenic IHC practice). Titrate around the catalog image concentration of 2.5 µg/mL without treating that image as proof of an optimal concentration for every specimen (A08612-1 caption). Check whether signal follows the expected cytoplasmic or perinuclear distribution rather than tissue folds, damaged edges or precipitate, and compare with the no primary control (UniProt Q8N5B7 subcellular annotation; standard IHC practice).
How should I score CERS5 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored cell population and compartment before analysis, then apply the same threshold, chromogen development time and imaging settings to every section (standard IHC practice). Report the percentage of positive cells and an H score calculated as the sum of each intensity category’s percentage times its category value; state the category scale used (standard IHC quantification practice). Alternatively, count positive cells per mm² within a prespecified viable tissue region and normalize to that region’s measured area or total eligible cells (standard IHC quantification practice). Keep cell types separate because reported CERS5 staining differs among Sertoli cells, cortical glia and cardiomyocytes (HPA: High in Sertoli cells; Medium in cerebral cortex glial cells; Not detected in cardiomyocytes).
When is a CERS5 positive signal convincing rather than artefactual?
A convincing result has reproducible cellular staining with a plausible cytoplasmic or perinuclear pattern and a clean no primary control (UniProt Q8N5B7 subcellular annotation; standard IHC practice). Use cell identity as a cross check: high staining is reported in Sertoli cells, medium staining in cerebral cortex glia, and no detection in cardiomyocytes (HPA tissue IHC). Treat strong signal confined to section edges, folds or necrotic areas as suspect, and check for endogenous peroxidase signal when using chromogenic detection (standard IHC practice). Nucleolar tissue staining has also been reported, so resolve an unexpected nuclear pattern with controls and independent validation before rejecting or accepting it (HPA tissue IHC; standard IHC practice).
Boster reagents
Best CERS5 / Ceramide synthase 5 IHC Antibodies
The catalog includes CERS5 antibodies with human brain IHC or IF images and listed human, mouse, and rat reactivity (catalog applications, reactivity, and image captions).
Real IHC data
Anti-LASS5 CERS5 Antibody
Cat # A08612-1
Real IF data
Anti-LASS5 CERS5 Antibody
Cat # A08612
A08612-1 lists IHC-P and IF, with images of human brain tissue IHC at 2.5 μg/mL and human brain cells IF at 20 μg/mL (A08612-1 applications and image captions). A08612 lists IF and shows human brain tissue IF at 20 μg/mL (A08612 applications and image caption).
Which to pick: For paraffin-section tissue IHC, choose A08612-1: it lists IHC-P and shows human brain tissue IHC at 2.5 μg/mL; the fixative is unreported (A08612-1 applications and image caption). For IF, A08612 shows human brain tissue staining; A08612-1 also lists IF and shows human brain cells, but neither catalog entry separately lists ICC (A08612 and A08612-1 applications and image captions). Both list human, mouse, and rat reactivity, while their supplied images show human samples; clonality is unreported for both (catalog reactivity, image captions, and clone fields).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8N5B7 (CERS5_HUMAN, Ceramide synthase 5).
- Human Protein Atlas. CERS5 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. CERS5 subcellular location (ICC-IF): Mainly localized to the nuclear membrane. In addition localized to the endoplasmic reticulum and primary cilium..
- Human Protein Atlas. CERS5 antibody validation summary (2 antibodies).
- High expression of ceramide synthase 5 predicts a poor prognosis in gastric cancer. Translational cancer research 2022 — PMC9552061.
- High CerS5 expression levels associate with reduced patient survival and transition from apoptotic to autophagy signalling pathways in colorectal cancer. The journal of pathology. Clinical research 2015 — PMC4858121.
- Ceramide Synthase 5 Deficiency Aggravates Dextran Sodium Sulfate-Induced Colitis and Colon Carcinogenesis and Impairs T-Cell Activation. Cancers 2020 — PMC7409364.
- Comprehensive profiling of lipid metabolic reprogramming expands precision medicine for HCC. Hepatology (Baltimore, Md.) 2025 — PMC11902616.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:16541075 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CERS5 (Ceramide synthase 5). Expected localisation ER membrane (UniProt); cytoplasmic/nucleolar tissue staining (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A08612-1 at 2.5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Sertoli cells); negative Heart muscle. 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 A08612-1); 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-CERS5, 2.5 μg/mL (datasheet A08612-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A08612-1); Caveat: Nucleolar staining warrants validation against the ER expectation (HPA tissue IHC; UniProt); Isoform / epitope: 2 isoforms; check luminal versus cytoplasmic epitope coverage (UniProt); Regulation: PAQR4 regulates CERS5 stability (UniProt). Evidence: UniProt Q8N5B7, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nuclear membrane. In addition localized to…, Datasheet A08612-1, PMC9552061, PMC4858121. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CERS5/immunohistochemistry-ihc-guide.