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CLUH / Clustered mitochondria protein homolog · IHC design guide
Design Immunohistochemistry for CLUH
Plan CLUH staining in paraffin sections around the cytoplasmic tissue pattern reported by HPA (HPA tissue IHC). The catalog antibody has an IHC dilution range of 1:50–1:200 (datasheet); interpret staining cautiously because HPA rates the tissue evidence uncertain (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CLUH 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 | Cytoplasmic staining in most tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in most tissues, including smooth muscle (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Breast+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 paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Antibody staining and RNA expression have low consistency (HPA tissue IHC) | |
| Regulation | Low tissue RNA specificity; no regulator established (HPA tissue RNA) | |
| Isoform / epitope | One full-length chain; no isoforms reported (UniProt) |
Section 1
Recommended CLUH IHC & IF Protocols
The catalog antibody IHC-P protocol (datasheet) is accompanied by a published CLUH protocol for human colonic biopsy sections (PMC10393232).
| Sample | Paraffin-embedded human placenta tissue; fixative not specified (datasheet A11184) |
| Fixation | Image fixative and duration unreported (datasheet A11184); 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-CLUH, 1:50-1:200 (datasheet A11184) |
| 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 | CLUH-positive staining in myoepithelial cells of breast (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in most tissues, including smooth muscle. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page antigen retrieval). The published protocol does not specify retrieval (PMC10393232: Methods).
Section 2
What Is the Expected CLUH Staining Pattern?
CLUH is a cytoplasmic protein with no transmembrane segment; a fraction occurs in cytoplasmic granules near mitochondria (UniProt O75153). In paraffin section IHC, expect cytoplasmic staining across many tissues, including smooth muscle, with medium staining reported in selected cell populations such as esophageal squamous epithelial cells and cardiomyocytes (HPA tissue IHC). Treat the pattern as provisional: HPA rates tissue IHC reliability Uncertain because staining and RNA expression have low consistency (HPA tissue IHC).
| Cytoplasmic chromogen in esophageal squamous epithelial cells or cardiomyocytes, with recognizable cell boundaries. | This fits the reported medium staining in those populations and the cytoplasmic localization of CLUH (HPA tissue IHC; UniProt O75153). Score intensity within the identified cell population, while allowing for the uncertain tissue IHC reliability (HPA tissue IHC). |
| Predominantly nuclear, membranous, or extracellular staining, with little cytoplasmic signal. | This is discordant with the expected cytoplasmic pattern and the absence of a transmembrane segment (UniProt O75153). Check morphology and detection controls before interpreting it as CLUH; the additional nucleolar signal reported by ICC-IF is uncertain and does not establish a nuclear IHC pattern (HPA subcellular). |
| Strong signal in adipocytes or adrenal glandular cells, especially when expected cytoplasmic populations are unstained. | Those populations are listed as not detected in HPA tissue IHC (HPA tissue IHC). Consider cross-reactivity or endogenous detection activity, then assess a no-primary control; HPA's uncertain reliability means these populations are comparators, not definitive negatives (HPA tissue IHC; general IHC practice). |
| Uniform haze across cells and surrounding tissue, obscuring compartment boundaries. | The pattern cannot support a cytoplasmic CLUH call (UniProt O75153). In chromogenic IHC, incomplete blocking, excess antibody, or detection background can cause diffuse staining; compare a no-primary control and optimize the general IHC workflow before scoring (general IHC practice). |
| No signal in an intact section containing esophageal squamous epithelial cells or cardiomyocytes. | Both populations have reported medium IHC staining, so absence warrants investigation (HPA tissue IHC). Verify tissue identity and assay controls, then check retrieval and detection conditions as general IHC steps; HPA's uncertain reliability prevents treating a single negative section as proof of CLUH absence (HPA tissue IHC; general IHC practice). |
💡Expected CLUH appearanceCall a result positive when a defined population shows cytoplasmic chromogen, potentially granular, with medium staining in reported esophageal squamous epithelial cells or cardiomyocytes; isolated nuclear, membrane, extracellular, or diffuse tissue-wide color is suspect (UniProt O75153; HPA tissue IHC; general IHC practice).
| Cell population and tissue context | HPA reports cytoplasmic expression in most tissues, but staining varies by cell population: esophageal squamous epithelial cells and cardiomyocytes are medium, whereas adipocytes are not detected (HPA tissue IHC). Use the named cells when comparing sections; avoid assigning one intensity to every cell in a tissue (HPA tissue IHC). |
| Localization and protein topology | UniProt places CLUH in the cytoplasm and cytoplasmic granules, with a fraction close to mitochondria, and lists no transmembrane segment (UniProt O75153). Proximity to mitochondria does not establish staining inside mitochondria; compartment calls should follow the visible cytoplasmic pattern (UniProt O75153). |
| Protein processing | UniProt lists one chain spanning residues 1–1309, with no signal peptide or propeptide annotated (UniProt O75153). The supplied record therefore offers no basis for expecting a secreted, shed, or separately processed CLUH staining compartment (UniProt O75153). |
| Strength of IHC evidence | HPA rates tissue IHC Uncertain because antibody staining and RNA expression have low consistency; its IHC entry for HPA044346 is also Uncertain (HPA tissue IHC; HPA antibodies). Treat an unexpected pattern as a finding to check with morphology and controls, not as established CLUH distribution (HPA tissue IHC; general IHC practice). |
| IF/ICC Q: what pattern can be expected? | HPA reports vesicles as the approved main ICC-IF location; its additional nucleoli fibrillar-center location is uncertain, with images listed for A-549, CACO-2, and U2OS (HPA subcellular). HPA031505 is Approved for ICC but has no IHC status listed, so that IF evidence does not validate paraffin-section IHC (HPA antibodies). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected cytoplasmic cells are blank while the section's detection control also fails. | The assay may have a general detection or reagent problem; this pattern cannot determine whether CLUH is absent (general IHC practice). | Restore the detection control first, then repeat staining and reassess an HPA-reported medium population such as cardiomyocytes (general IHC practice; HPA tissue IHC). |
| Expected cells are blank, but the detection control works. | Possible issues include antibody performance or an unsuitable retrieval condition; no CLUH-specific retrieval or fixation sensitivity is established by the supplied UniProt and HPA records. | Verify the antibody's documented IHC use and optimize retrieval with controlled comparisons as general IHC practice; do not infer CLUH absence from this result alone (general IHC practice; HPA tissue IHC: Uncertain). |
| The no-primary control develops chromogen. | Endogenous enzyme activity or another detection-system background source can produce color without CLUH antibody binding (general IHC practice). | Check the detection chemistry, apply the appropriate endogenous-activity block, and repeat the no-primary control before scoring tissue staining (general IHC practice). |
| Color forms a diffuse haze or obscures cytoplasmic boundaries. | Nonspecific binding or excess primary or detection reagent can obscure the compartment expected for CLUH (general IHC practice; UniProt O75153). | Adjust blocking, washes, and antibody concentration according to the assay controls; score only when cell boundaries and cytoplasmic localization can be assessed (general IHC practice; UniProt O75153). |
| Strong nuclear or cell-surface color dominates an otherwise intact section. | That distribution conflicts with the UniProt cytoplasmic location and lack of a transmembrane segment; HPA's additional nucleolar ICC-IF location is uncertain (UniProt O75153; HPA subcellular). | Compare the no-primary control and cell morphology, then seek independently supported cytoplasmic staining before assigning the signal to CLUH (general IHC practice; UniProt O75153). |
| A reported negative population stains strongly, or reported positive populations vary between sections. | Background or cell-identification error is possible; HPA also rates the tissue pattern Uncertain, so its listed levels are imperfect comparators (HPA tissue IHC; general IHC practice). | Confirm the cell population and section quality, compare positive and negative populations on the same run, and document the discrepancy rather than forcing an HPA-based positive or negative call (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing CLUH staining
A11184 · IHCImmunohistochemistry of paraffin-embedded human placenta using CLUH antibody at dilution of 1:100 .Perform microwave antigen retrieval with
A11184 · IHCImmunohistochemistry of paraffin-embedded human stomach using CLUH antibody at dilution of 1:100 .Perform microwave antigen retrieval with 1
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000132361-CLUH/tissue/cerebral+cortex#imid_11906475
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000132361-CLUH/tissue/colon#imid_11906409
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000132361-CLUH/tissue/liver#imid_11906410
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000132361-CLUH/tissue/kidney#imid_11906395
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA031505; green = CLUH, blue = nucleus, red = microtubules.
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA031505; green = CLUH, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CLUH IHC & IF
🧪Run breast first and expect staining in myoepithelial cells (HPA: Medium in breast myoepithelial cells). Use adipose tissue as the negative comparator (HPA: Not detected in adipocytes); on the breast slide, treat other cell types as background references only after independently confirming they lack CLUH.
Positive control tissue: Breast (Myoepithelial cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CLUH in A-549, CACO-2, U2OS, with annotated localisation: Vesicles (approved) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and host-species-matched controls: isotype-matched immunoglobulin for a monoclonal primary or nonimmune IgG for a polyclonal primary. Use CLUH knockout tissue or cells as a biological negative, or an immunizing-peptide block if the peptide is available; for chromogenic breast IHC, quench endogenous peroxidase and compare residual signal with the no-primary slide.
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the fixative in the selected A11184 paraffin-section caption is unreported (catalog caption). That caption uses microwave retrieval in 10 mM PBS at pH 7.2 before staining placenta at 1:100, but retrieval dependence in breast is unreported (catalog caption). The supplied evidence does not establish that frozen sections or IF are easier; in breast, use the counterstain and tissue morphology to distinguish myoepithelial signal from adjacent cells.
HPA tissue IHC evidence for CLUH
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — 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 |
|---|---|---|---|---|
| Breast | Myoepithelial cells | Medium | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | Endothelial cells | Medium | Protein (IHC) | HPA → |
| Colon | Endothelial cells | Medium | Protein (IHC) | HPA → |
| Duodenum | Glandular 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 → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CLUH IHC Tips
Use the paraffin-section image as a starting reference, and interpret CLUH staining against its reported cytoplasmic distribution and the uncertain tissue-IHC reliability (A11184 caption; UniProt O75153; HPA tissue IHC).
Which retrieval conditions should I try first for CLUH in paraffin sections?
Use citrate buffer at pH 6.0 for heat-induced retrieval at 95–98 °C for 20 min (page IHC protocol). If staining is weak, compare a matched section using microwave retrieval in 10 mM PBS, pH 7.2, the method reported for the catalog antibody in paraffin-embedded human placenta (A11184 caption). Hold antibody concentration, detection, and development time constant across sections so the comparison isolates retrieval (standard IHC practice). The caption reports 1:100 antibody dilution but does not report a fixative, so it cannot establish how fixation changes retrieval performance (A11184 caption).
How should I assess whether fixation is masking CLUH staining?
Target-specific fixation sensitivity is unknown because the supplied CLUH tissue-IHC caption does not identify its fixative (A11184 caption). Process matched paraffin sections with a consistent documented fixation history, then compare retrieval and staining under identical conditions (standard IHC practice). Include a reference section from the same processing batch and record fixation duration, retrieval conditions, and chromogen development time for each run (standard IHC practice). If signal differs between batches, treat fixation as one possible variable rather than attributing the change to CLUH biology; neither the reported cytoplasmic location nor phosphorylation sites establish fixation sensitivity (UniProt O75153; standard IHC practice).
What staining pattern fits CLUH, and how should puncta be evaluated?
Assess CLUH primarily in the cytoplasm: UniProt reports cytoplasmic granules and a fraction near mitochondria and tyrosinated tubulin (UniProt O75153). HPA describes cytoplasmic tissue staining, while its cell imaging places the main signal in vesicles (HPA tissue IHC; HPA subcellular). Examine diffuse and punctate cytoplasmic signal within identified cells, then compare the pattern with a matched negative control and tissue morphology (standard IHC practice). Do not call puncta mitochondrial solely from chromogenic proximity: CLUH has no transmembrane segment, and the supplied localisation evidence does not make every punctum a mitochondrion (UniProt O75153).
What can I conclude about CLUH epitope accessibility from the supplied sequence record?
The supplied record lists one 1–1309 chain and 0 annotated isoforms, but gives no antibody epitope coordinates (UniProt O75153). It therefore cannot establish whether the catalog antibody recognizes the Clu domain at residues 335–577 or any listed phosphoserine site (UniProt O75153; A11184 caption). Compare adjacent paraffin sections under the same detection conditions when testing retrieval changes, and judge accessibility by specific cellular staining against controls (standard IHC practice). A changed signal after retrieval does not, by itself, identify an epitope or demonstrate modification-dependent binding (standard IHC interpretation).
How can I evaluate CLUH by multiplex IF alongside the chromogenic IHC result?
For a separate IF experiment, pair CLUH with a marker of the cell population being assessed, such as a validated trophoblast marker when examining the placenta shown in the IHC caption (A11184 caption; standard IF practice). Choose fluorophores and imaging channels after checking unstained tissue autofluorescence, and include single-label controls to assess channel bleed-through (standard IF practice). Because CLUH is cytoplasmic and has no transmembrane segment, test permeabilisation suitable for an intracellular epitope while recognizing that this antibody’s epitope is unspecified (UniProt O75153; standard IF practice). HPA cell images report vesicular signal, but they do not validate this catalog antibody for IF (HPA subcellular; A11184 caption).
How can I separate nonspecific brown staining from CLUH signal?
Run a no-primary control and inspect regions with endogenous pigment or enzyme activity before calling faint brown signal positive (standard chromogenic IHC practice). Apply a peroxidase block for peroxidase-based detection, then compare DAB development times across matched sections; these are general workflow steps, not CLUH-specific evidence (standard IHC practice). If background persists, assess blocking, antibody dilution, wash conditions, and section edges one variable at a time (standard IHC practice). The catalog image reports 1:100 antibody dilution in paraffin-embedded placenta, which is a documented starting condition rather than a demonstrated optimum for every tissue (A11184 caption).
How should I score CLUH staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and cytoplasmic compartment before scoring, because HPA reports cytoplasmic tissue expression and UniProt places CLUH in the cytoplasm (HPA tissue IHC; UniProt O75153). For chromogenic sections, record the percentage of positive cells and staining intensity, or calculate an H-score from the same predefined cell population (standard IHC practice). Normalize counts to the number of eligible cells; if measuring positive-cell density, report cells per mm² of analyzable tissue (standard IHC practice). Keep retrieval, antibody dilution, detection, and image thresholds consistent across groups, and report HPA’s uncertain tissue-IHC reliability when interpreting differences (standard IHC practice; HPA tissue IHC).
When should I question an apparent CLUH-positive IHC result?
A convincing result should fit a cytoplasmic pattern in intact cells and remain distinct from staining in the no-primary control (UniProt O75153; standard IHC practice). Re-examine isolated nuclear staining, edge-only signal, necrotic regions, and deposits associated with endogenous enzyme activity before assigning CLUH positivity (UniProt O75153; standard IHC practice). HPA reports low consistency between antibody staining and RNA expression, so its tissue examples are context rather than proof for a new specimen (HPA tissue IHC). In placenta, compare the observed cells with the reported low trophoblastic staining and the catalog image, while accounting for their differing evidence and the caption’s unreported fixative (HPA tissue IHC; A11184 caption).
Boster reagents
Best CLUH / Clustered mitochondria protein homolog IHC Antibodies
Anti-CLUH A11184 has IHC images from paraffin-embedded human placenta and stomach (catalog image captions); IF/ICC is listed for human and mouse, with no IF image supplied (catalog applications, reactivity, image alts).
Real IHC data
Anti-CLUH Antibody
Cat # A11184
A11184 will render with an IHC figure from paraffin-embedded human placenta at 1:100 after microwave retrieval in 10 mM PBS, pH 7.2 (A11184 IHC image caption). A second caption documents human stomach under the same conditions; IF/ICC is listed, but no IF image is supplied (A11184 IHC image captions; catalog applications and IF image alts).
Which to pick: Choose A11184 for paraffin-section tissue IHC because its own captions document staining in human placenta and stomach; the fixative is unreported (A11184 IHC image captions). A11184 is also the listed IF/ICC option, with a 1:50–1:200 dilution range, though no IF image is supplied (catalog applications, dilution, IF image alts). For human or mouse samples, A11184 is the listed rabbit polyclonal option; the pictured IHC samples are human (catalog host, dilution_raw, reactivity; A11184 IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O75153 (CLU_HUMAN, Clustered mitochondria protein homolog).
- Human Protein Atlas. CLUH tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. CLUH subcellular location (ICC-IF): Mainly localized to vesicles. In addition localized to the nucleoli fibrillar center..
- Human Protein Atlas. CLUH antibody validation summary (2 antibodies).
- CLUH functions as a negative regulator of inflammation in human macrophages and determines ulcerative colitis pathogenesis. JCI insight 2023 — PMC10393232.
- Clueless/CLUH regulates mitochondrial fission by promoting recruitment of Drp1 to mitochondria. Nature communications 2022 — PMC8948191.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:17974005 — UniProt-cited evidence.
- PubMed PMID:9734811 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CLUH (Clustered mitochondria protein homolog). Expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A11184 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Breast (Myoepithelial cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human placenta tissue; fixative not specified (datasheet A11184); 2. Fixation: Image fixative and duration unreported (datasheet A11184); 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-CLUH, 1:50-1:200 (datasheet A11184); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Antibody staining and RNA expression have low consistency (HPA tissue IHC); Isoform / epitope: One full-length chain; no isoforms reported (UniProt); Regulation: Low tissue RNA specificity; no regulator established (HPA tissue RNA). Evidence: UniProt O75153, HPA tissue IHC · Uncertain, HPA ICC-IF · Mainly localized to vesicles. In addition localized to the nucleoli…, Datasheet A11184, PMC10393232. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CLUH/immunohistochemistry-ihc-guide.