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CLUH / Clustered mitochondria protein homolog · IHC design guide
Design Immunohistochemistry for CLUH
Plan chromogenic CLUH IHC in paraffin sections around the cytoplasmic tissue pattern (HPA tissue IHC). Use the catalog antibody's 1:50–1:200 IHC range (datasheet), and interpret staining cautiously because its agreement with RNA expression is low (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 tissue staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic signal 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 | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A11184) | |
| Caveat | Staining and RNA expression show low consistency (HPA tissue IHC) | |
| Regulation | No expression regulator annotated (UniProt) | |
| Isoform / epitope | No annotated isoforms; one 1–1309 chain (UniProt) |
Section 1
Recommended CLUH IHC & IF Protocols
The catalog antibody protocol and a published human colonic biopsy protocol provide starting points for CLUH IHC (PMC10393232: methods).
| 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?
Expect chiefly cytoplasmic CLUH staining in many tissues, including smooth muscle (HPA: tissue IHC); reported medium staining includes breast myoepithelial cells and heart cardiomyocytes (HPA: tissue IHC). CLUH is cytoplasmic, can occur in cytoplasmic granules near mitochondria, and has no transmembrane segment (UniProt O75153). Interpret tissue staining cautiously: HPA rates its IHC profile Uncertain because antibody staining and RNA expression show low consistency (HPA: tissue IHC).
| Cytoplasmic staining in breast myoepithelial cells or heart cardiomyocytes. | This matches reported medium staining in those cells (HPA: tissue IHC) and the cytoplasmic assignment (UniProt O75153). Score the stained cell population and intensity alongside a negative detection control (general IHC practice); the matching pattern alone does not resolve HPA’s Uncertain IHC reliability (HPA: tissue IHC). |
| Predominantly crisp nuclear or cell-surface staining, with little cytoplasmic signal. | That distribution conflicts with CLUH’s cytoplasmic location and lack of a transmembrane segment (UniProt O75153). Suspect nonspecific staining or a detection artefact; check the negative detection control and reassess the antibody result (general IHC practice). Do not use an isolated nuclear signal as evidence of CLUH. |
| Signal appears mainly in cells unexpected for the selected tissue. | Compare the cells with that tissue’s HPA annotation before calling them unexpected: HPA reports medium staining in several distinct cell populations and low staining in lung macrophages (HPA: tissue IHC). If the pattern still conflicts, consider antibody cross-reactivity or endogenous detection activity and inspect controls (general IHC practice). |
| Diffuse chromogen covers stroma, empty spaces, or many cell compartments. | Such spread obscures the cell-restricted cytoplasmic pattern expected for CLUH (UniProt O75153; HPA: tissue IHC). Examine a negative detection control, blocking, washes, and chromogen development before assigning positive cells (general IHC practice). |
| No signal in breast myoepithelial cells or heart cardiomyocytes. | HPA reports medium staining in these populations, so absence warrants a run check (HPA: tissue IHC). Verify tissue preservation, retrieval and detection controls under the laboratory’s IHC workflow (general IHC practice). HPA’s Uncertain reliability means either population is a reference pattern, not a guaranteed positive control (HPA: tissue IHC). |
💡Expected CLUH appearanceA credible positive shows cell-associated cytoplasmic chromogen, potentially granular, with medium staining in reported breast myoepithelial cells or heart cardiomyocytes (UniProt O75153; HPA: tissue IHC); isolated nuclear, surface, or diffuse acellular color warrants investigation (general IHC practice).
| Compartment and topology | CLUH is cytoplasmic, can form cytoplasmic granules, and has no transmembrane segment (UniProt O75153). A fraction lies close to mitochondria (UniProt O75153); proximity does not make exclusive mitochondrial staining a required IHC pattern. |
| Tissue evidence | HPA reports cytoplasmic expression in most tissues, but marks its tissue IHC profile Uncertain because staining has low consistency with RNA expression (HPA: tissue IHC). Its listed Not detected populations are comparison points, not proof that every negative cell lacks CLUH. |
| Antibody validation | The listed IHC antibody has an Uncertain IHC status, while the separately listed ICC antibody has Approved ICC status (HPA: antibodies HPA044346, HPA031505). Those status labels apply to their respective assays; ICC approval does not validate an IHC result. |
| Retrieval and detection | Paraffin IHC commonly uses antigen retrieval and chromogenic detection controls (general IHC practice). The supplied HPA and UniProt records give no CLUH-specific retrieval condition, dilution, or fixation-sensitivity result; use the IHC-validated antibody’s supplied protocol if available. |
| IF/ICC Q&A: should puncta or nucleoli be expected? | HPA places CLUH mainly in vesicles by ICC-IF, with Approved status, and additionally reports a nucleoli fibrillar-center location with Uncertain status (HPA: subcellular). Treat that as IF/ICC context; it does not establish a required nucleolar pattern in paraffin IHC. |
| Situation | Likely cause | Next action |
|---|---|---|
| The reference cell population stains weakly or not at all. | Retrieval, antibody incubation, or detection may have failed (general IHC practice); HPA’s Uncertain IHC reliability also limits how strongly absence can be interpreted (HPA: tissue IHC). | Check run controls, tissue quality, the applied retrieval workflow, and reagent performance before judging CLUH negative (general IHC practice). Compare with the reported cell-specific pattern, not whole-tissue darkness (HPA: tissue IHC). |
| Chromogen appears in the negative detection control. | Background from detection chemistry or endogenous activity is possible (general IHC practice). The staining cannot be attributed to CLUH from this control result alone. | Correct the detection or blocking conditions and repeat the control (general IHC practice). Interpret sample staining only after control background is acceptably low. |
| Most structures show uniform brown haze. | Excess antibody signal, incomplete washing, or overdevelopment can obscure cell boundaries (general IHC practice). This appearance does not support the reported cytoplasmic pattern (HPA: tissue IHC). | Review antibody incubation, wash steps, and chromogen development using appropriate run controls (general IHC practice); then rescore identifiable cells and compartments. |
| Only nuclei or cell borders are sharply positive. | The dominant pattern conflicts with the cytoplasmic location and lack of a transmembrane segment (UniProt O75153). Nonspecific binding or detection artefact is possible (general IHC practice). | Inspect negative detection controls and repeat with validated IHC conditions (general IHC practice). Record the compartment discrepancy instead of counting those cells as CLUH-positive. |
| A listed Not detected cell population stains. | HPA lists specific populations, such as adipocytes in adipose tissue, as Not detected, yet rates the overall tissue IHC profile Uncertain (HPA: tissue IHC). A discrepancy alone cannot establish cross-reactivity. | Confirm cell identity and compartment, examine negative detection controls, and compare another documented tissue population if available (general IHC practice; HPA: tissue IHC). |
| ICC-IF shows vesicles, but the paraffin IHC slide looks broadly cytoplasmic. | Vesicles are the Approved main ICC-IF location, whereas HPA summarizes tissue IHC as cytoplasmic (HPA: subcellular; HPA: tissue IHC). The two observations come from different assays. | Score the IHC slide against the tissue IHC cell and compartment pattern (HPA: tissue IHC). Use the separate IF/ICC guide for IF interpretation; do not infer an IHC protocol from the ICC-IF images. |
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 look for CLUH staining in myoepithelial cells (HPA: Medium in breast myoepithelial cells). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes); on the breast slide, cells without specific staining should remain at background rather than be assigned a presumed negative cell type.
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 primary-host-species- and clonality-matched isotype controls, plus a CLUH knockout specimen or a peptide block if the immunizing peptide is available (standard IHC practice). For chromogenic detection, quench endogenous peroxidase and, if using avidin–biotin detection, control for endogenous biotin in breast tissue (standard IHC practice).
⚠️Feasibility: A CLUH-specific fixation window or fixation effect is unreported in the supplied evidence, and the selected A11184 paraffin-section caption does not state a fixative (A11184 tissue-IHC caption: fixative unreported). The caption reports microwave retrieval in 10 mM PBS, pH 7.2, for placenta at 1:100; it does not establish whether retrieval is required for breast tissue (A11184 tissue-IHC caption). The supplied evidence does not establish whether frozen sections or IF are easier than paraffin IHC, or identify a breast-specific artefact (HPA: breast myoepithelial cells Medium; HPA: ICC-IF images in A-549, CACO-2 and U2OS).
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
Troubleshoot chromogenic CLUH IHC in paraffin sections by checking retrieval, cytoplasmic localisation, controls and cell-specific scoring before interpreting staining.
What retrieval conditions should I start with for CLUH in paraffin sections?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 minutes (page retrieval rule: cytoplasmic antigen). If staining is weak, compare a matched section using microwave retrieval in 10 mM PBS, pH 7.2, the condition shown for paraffin-embedded human placenta with antibody A11184 (A11184 tissue-IHC caption). Keep antibody dilution, detection and imaging conditions identical across sections so the retrieval comparison is interpretable (standard IHC practice). Score cytoplasmic staining within comparable cell populations, since CLUH is reported in the cytoplasm and cytoplasmic granules (UniProt O75153 localisation).
Could fixation explain weak CLUH staining in my paraffin sections?
Target-specific CLUH sensitivity to fixation is unknown from the supplied evidence; the A11184 placenta caption identifies paraffin embedding but does not state a fixative (A11184 tissue-IHC caption). Record the actual fixative and fixation duration for each specimen, then compare sections processed with the same retrieval and detection conditions (standard IHC practice). Excessive fixation can reduce epitope accessibility in IHC generally, so a controlled fixation series can test this possibility without assuming a CLUH-specific effect (standard IHC practice). Include a known staining control alongside each batch and evaluate cytoplasmic signal in equivalent cells before changing antibody concentration (UniProt O75153 localisation; standard IHC practice).
Where should convincing CLUH chromogenic staining appear?
Look first for cytoplasmic staining: CLUH is annotated in the cytoplasm and cytoplasmic granules, with a fraction near mitochondria and colocalising with tyrosinated tubulin (UniProt O75153 localisation). It has no transmembrane segment, so an isolated crisp membrane outline needs independent validation before assignment to CLUH (UniProt O75153 topology). HPA describes mainly vesicular localisation in cell images and lists nucleolar fibrillar-center localisation as uncertain; neither establishes a nuclear IHC scoring pattern (HPA subcellular). Compare the signal with cell morphology and a negative detection control, and document diffuse versus punctate cytoplasmic patterns separately (standard IHC practice).
How should epitope uncertainty affect antibody comparison?
The supplied record lists 0 isoforms and a single CLUH chain spanning residues 1–1309, so it provides no isoform-specific IHC interpretation (UniProt O75153 processing and isoforms). Its Clu domain spans residues 335–577, and recorded phosphoserines include positions 279, 281, 654, 664 and 723 (UniProt O75153 domains and modified residues). The antibody epitope is not supplied, so do not claim that retrieval exposes a particular domain or that phosphorylation causes a staining change (supplied antibody evidence). If two antibodies disagree, compare their documented immunogens when available and test matched sections with identical retrieval, detection and scoring (standard IHC practice).
How can I check a CLUH IHC pattern by multiplex immunofluorescence?
For a separate IF experiment, pair CLUH with a marker identifying the cell population being evaluated, such as a validated trophoblast marker in placenta; HPA reports low CLUH staining in trophoblastic cells (HPA tissue IHC). Choose fluorophores after inspecting unstained tissue autofluorescence, favouring channels with clear separation from that background, and include single-stain controls (standard IF practice). CLUH is cytoplasmic and has no transmembrane segment, so use permeabilisation suitable for an intracellular epitope and verify that cell morphology remains intact (UniProt O75153 localisation and topology; standard IF practice). Treat the A11184 paraffin tissue caption as IHC evidence only; it does not establish IF fixation or permeabilisation conditions (A11184 tissue-IHC caption).
What should I check when CLUH staining is widespread or muddy?
First compare a no-primary control with the stained section to identify detection-system background, then inspect unstained tissue for endogenous pigment (standard chromogenic IHC practice). If using peroxidase detection, include a peroxidase block and distinguish residual endogenous enzyme activity from DAB signal with the control (standard chromogenic IHC practice). Titrate the primary antibody and strengthen blocking or washing while keeping retrieval constant, because excess reagent can obscure cytoplasmic detail (standard IHC practice). Widespread cytoplasmic staining alone is not proof of specificity: HPA reports expression in most tissues but rates its tissue IHC reliability uncertain because staining and RNA data have low consistency (HPA tissue IHC).
How should I quantify CLUH across differently composed tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and cytoplasmic scoring compartment before review, since HPA reports different staining levels across named cell types (HPA tissue IHC). Record the percentage of positive target cells and staining intensity, then calculate an H-score as the sum of intensity category multiplied by its percentage, using a consistent category scale (standard IHC scoring practice). For spatial comparisons, report positive-cell density per mm² of viable, annotated tissue and exclude folds, edges and necrosis (standard IHC scoring practice). Normalise within the same cell type or to its total counted cells, and keep retrieval, illumination and threshold settings consistent across cases (standard IHC practice).
How can I distinguish credible CLUH staining from artefact?
Credible staining should follow a cytoplasmic or granular pattern in intact cells, consistent with CLUH localisation, rather than appear only as a sharp cell-membrane rim (UniProt O75153 localisation and topology). Check the identity of stained cells: HPA reports medium staining in heart cardiomyocytes and low staining in placental trophoblastic cells, while its overall tissue IHC reliability is uncertain (HPA tissue IHC). Treat staining confined to section edges, folds or necrotic areas as suspect, and compare it with adjacent intact tissue (standard IHC practice). A no-primary control and adequate peroxidase block help identify detection background or endogenous enzyme signal before a DAB-positive area is called CLUH (standard chromogenic IHC practice).
Boster reagents
Best CLUH / Clustered mitochondria protein homolog IHC Antibodies
Anti-CLUH A11184 has human placenta and stomach paraffin-section IHC images (catalog IHC image captions). IF/ICC and Human/Mouse reactivity are listed, but no IF image is provided (catalog applications/reactivity/IF image alts).
Real IHC data
Anti-CLUH Antibody
Cat # A11184
The A11184 card shows human placenta IHC at 1:100 after microwave retrieval in 10 mM PBS, pH 7.2 (catalog card figure caption). A second catalog image documents the same conditions in human stomach; IF/ICC and Human/Mouse reactivity are listed without an IF image (catalog IHC image captions/applications/reactivity/IF image alts).
Which to pick: Choose A11184 for paraffin-section tissue IHC; its own captions document staining in human placenta and stomach (catalog IHC image captions). For IF/ICC or Mouse samples, A11184 is the listed rabbit polyclonal option, although the supplied images show human IHC only (catalog applications/reactivity/dilution_raw/IHC image captions/IF image alts). The paraffin-section captions do not report the fixative (catalog 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 tissue staining (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: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A11184); Caveat: Staining and RNA expression show low consistency (HPA tissue IHC); Isoform / epitope: No annotated isoforms; one 1–1309 chain (UniProt); Regulation: No expression regulator annotated (UniProt). 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.