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CHCHD2 / Coiled-coil-helix-coiled-coil-helix domain-containing protein 2 · IHC design guide
Design Immunohistochemistry for CHCHD2
Use high-staining glandular or neuronal tissues as positive controls and smooth muscle as a negative comparator (HPA tissue IHC). This guide covers paraffin-section IHC planning and scoring of granular cytoplasmic CHCHD2 staining (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CHCHD2 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 | Observed granular cytoplasm (HPA tissue IHC); mainly mitochondrial intermembrane space (UniProt) | |
| Staining pattern | Granular cytoplasm in glandular and neuronal cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A06266-1) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Smooth muscle |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Expression regulation is not annotated (UniProt) | |
| Isoform / epitope | 0 isoforms annotated; intact 1–151 chain (UniProt) |
Section 1
Recommended CHCHD2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by a published human renal cell carcinoma tissue microarray protocol (PMC6313053).
| Sample | Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet A06266-1) |
| Fixation | Image fixative and duration unreported (datasheet A06266-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A06266-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A06266-1) |
| Primary antibody | Rabbit anti-CHCHD2, 1:50 recommended; image 1:200 (datasheet A06266-1) |
| Primary incubation | Overnight at 4 °C (datasheet A06266-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A06266-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CHCHD2-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression with a granular pattern. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet A06266-1); the published protocol does not specify retrieval (PMC6313053).
Section 2
What Is the Expected CHCHD2 Staining Pattern?
CHCHD2 is mainly mitochondrial, in the intermembrane space, with a nuclear location also annotated (UniProt Q9Y6H1). In paraffin tissue IHC, expect granular cytoplasmic staining, including strong staining in several glandular and neuronal cell populations (HPA tissue IHC). HPA rates the tissue staining Approved, with medium consistency against RNA data (HPA tissue IHC). CHCHD2 has no transmembrane segment (UniProt Q9Y6H1 topology).
| Granular cytoplasmic stain in colon or duodenal glandular cells. | This matches HPA's general granular cytoplasmic profile and High staining in those cells (HPA tissue IHC). Score the cells and compartment together; a positive tissue section alone does not show that every stained structure is CHCHD2. |
| Signal is confined to a smooth cell border or extracellular material. | That distribution conflicts with the mainly mitochondrial location (UniProt Q9Y6H1; HPA subcellular ICC-IF). Review it as a possible artefact. Nuclear signal alone needs more caution: UniProt also annotates a nuclear location (UniProt Q9Y6H1). |
| Strong stain in smooth muscle cells, with little stain in the expected glandular cells. | HPA reports smooth muscle cells as Not detected and several glandular populations as High (HPA tissue IHC). Suspect nonspecific staining or endogenous chromogenic activity, then compare controls; one discordant section cannot identify the cause. |
| Broad, even colour across cells and surrounding tissue obscures granules. | The slide cannot be scored against HPA's granular cytoplasmic profile (HPA tissue IHC). General IHC causes include nonspecific reagent binding or background from the detection system; assess these with appropriate negative controls. |
| No cellular signal in a well-preserved colon glandular region. | That is unexpected because colon glandular cells are High in HPA tissue IHC (HPA tissue IHC). Check the run and tissue before calling CHCHD2 absent; HPA's Approved rating has only medium consistency with RNA data (HPA tissue IHC). |
💡Expected CHCHD2 appearanceCall a convincing IHC positive when glandular or neuronal cells show clear granular cytoplasmic signal, potentially High in HPA-listed populations (HPA tissue IHC); uniform border, extracellular, or slide-wide colour is an atypical result to investigate (UniProt Q9Y6H1; HPA tissue IHC).
| Compartment and topology | CHCHD2 is mainly in the mitochondrial intermembrane space and has no transmembrane segment (UniProt Q9Y6H1). HPA describes tissue IHC as granular cytoplasmic and supported ICC-IF localisation as mitochondrial (HPA tissue IHC; HPA subcellular ICC-IF). |
| Tissue and cell selection | HPA lists High staining in colon and duodenal glandular cells and cortical and caudate neuronal cells; smooth muscle cells are Not detected (HPA tissue IHC). Compare the identified cell population, since an entire tissue can contain several cell types. |
| Strength of the evidence | Both listed antibodies are Approved for IHC, while HPA describes medium consistency between staining and RNA expression (HPA antibodies; HPA tissue IHC). Treat the recorded pattern as an interpretation benchmark, not proof of specificity in a new specimen. |
| IF/ICC Q: Where should CHCHD2 appear? | A: Mitochondria are the supported main location in HPA ICC-IF images; UniProt also annotates nucleus (HPA subcellular ICC-IF; UniProt Q9Y6H1). Use the separate IF/ICC guide for that application's workflow. |
| Target-specific fixation sensitivity | No CHCHD2-specific fixation effect is established by the supplied UniProt or HPA records. Record fixation and retrieval conditions for run comparison as general IHC practice, without interpreting a negative result as evidence of CHCHD2 epitope masking. |
| Situation | Likely cause | Next action |
|---|---|---|
| A positive tissue has no visible granular stain. | A failed IHC step, unsuitable detection conditions, or an unrepresentative region is possible; colon glandular cells are High in HPA tissue IHC (HPA tissue IHC). | Confirm that glandular cells are present, review the run's positive control, and check retrieval, antibody application, detection, and chromogen steps as general IHC practice. Avoid assigning a CHCHD2-specific fixation cause. |
| Smooth muscle stains as strongly as adjacent glandular cells. | That differs from HPA's Not detected smooth muscle result (HPA tissue IHC). Nonspecific binding or endogenous detection activity is possible in chromogenic IHC. | Compare the same cell types in control sections, including an appropriate negative detection control. Reassess background and detection conditions before scoring smooth muscle as CHCHD2-positive. |
| Stain is uniformly diffuse instead of granular. | Diffuse colour does not match the general granular cytoplasmic pattern (HPA tissue IHC). Excess background or poor contrast can obscure the cellular distribution in a chromogenic assay. | Inspect the negative control and counterstain, then review blocking, wash, antibody dilution, and chromogen development as general IHC practice. Score CHCHD2 only when the cellular pattern is interpretable. |
| Only nuclei stain in tissue IHC. | UniProt annotates nuclear CHCHD2, but HPA's general tissue pattern is granular cytoplasmic (UniProt Q9Y6H1; HPA tissue IHC). Nuclear-only staining therefore needs corroboration. | Check a HPA-listed High cell population for the expected cytoplasmic pattern and compare controls. Report nuclear-only staining as discordant with the usual tissue IHC profile, without declaring every nuclear signal false. |
| A strong outline or extracellular deposit is read as positive. | That pattern lacks the expected granular cytoplasmic distribution (HPA tissue IHC) and does not follow the mainly mitochondrial location (UniProt Q9Y6H1). | Identify the stained structure at higher magnification and compare a negative control. Exclude edge and extracellular colour from cellular scoring unless a supported CHCHD2 pattern is also visible. |
| Low hippocampal neuronal staining is mistaken for assay failure. | HPA records hippocampal neuronal cells as Low, while caudate and cerebral cortical neuronal cells are High (HPA tissue IHC). Regional differences can affect the expected intensity. | Verify the sampled region and compare an HPA-listed High population in the same run. Judge assay performance from the control and cellular pattern, not from hippocampal intensity alone. |
Experiment evidence
IHC & IF images showing CHCHD2 staining
A06266-1 · IHCIHC analysis of CHCHD2 using anti-CHCHD2 antibody (A06266-1). CHCHD2 was detected in a paraffin-embedded section of human lung cancer tissue
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000106153-CHCHD2/tissue/cerebral+cortex#imid_12258453
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000106153-CHCHD2/tissue/colon#imid_12265435
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000106153-CHCHD2/tissue/liver#imid_12265428
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000106153-CHCHD2/tissue/kidney#imid_12265421
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA027407; green = CHCHD2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA027407; green = CHCHD2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CHCHD2 IHC & IF
🧪Run colon first: its glandular cells should stain strongly (HPA: High in colon glandular cells). Run smooth muscle as the negative tissue (HPA: Not detected in smooth muscle cells); smooth muscle cells on the colon slide, where present, should show little or no signal (HPA: Not detected in smooth muscle cells).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Smooth muscle (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CHCHD2 in A-431, U-251MG, U2OS, with annotated localisation: Mitochondria (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and host- and isotype-matched rabbit IgG controls, plus CHCHD2 knockout tissue or a validated peptide-block control (standard IHC practice; catalog caption: rabbit primary). Block endogenous peroxidase before HRP/DAB detection, particularly where inflammatory cells are present in colon (standard IHC practice; catalog caption: HRP/DAB detection).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A06266-1 tissue-IHC caption does not report a fixative (catalog caption: fixative not stated). The demonstrated paraffin-section procedure uses heat retrieval in EDTA at pH 8.0, but retrieval dependency has not been established (catalog caption: EDTA pH 8.0). Neither frozen sections nor IF is shown to be easier; ICC-IF images support mitochondrial localization, which can guide interpretation of punctate staining (HPA: Mitochondria, supported).
HPA tissue IHC evidence for CHCHD2
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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CHCHD2 IHC Tips
Troubleshooting CHCHD2 staining in paraffin sections, with one note on translating the assay to IF/ICC.
How should I troubleshoot weak CHCHD2 staining after antigen retrieval?
Use heat-mediated EDTA at pH 8.0 for paraffin-section CHCHD2 IHC (datasheet A06266-1). In the selected lung cancer section, this preceded 10% goat-serum blocking and 1:200 primary antibody overnight at 4°C (datasheet A06266-1). If staining is weak, check paraffin removal, heating and cooling on adjacent sections before changing retrieval conditions (standard IHC practice). Compare any fallback retrieval condition on matched sections while holding antibody incubation and DAB development constant (standard IHC practice). Assess whether granular cytoplasmic signal improves without a matching rise in no-primary background (HPA tissue IHC; standard IHC practice).
Could fixation explain weak or uneven CHCHD2 staining?
The selected paraffin-section caption does not state a fixative, so CHCHD2-specific fixation sensitivity is unknown (datasheet A06266-1). Record each specimen’s fixative, fixation interval and processing history before comparing staining across sections (standard IHC practice). Use adjacent sections to compare processing conditions while keeping EDTA at pH 8.0 and the 1:200 overnight primary incubation consistent (datasheet A06266-1; standard IHC practice). Uneven preservation, detached tissue and edge-heavy staining warrant a processing check before biological scoring (standard IHC practice). The reported granular cytoplasmic pattern does not establish which fixative preserves this antigen best (HPA tissue IHC).
Is nuclear CHCHD2 staining plausible alongside granular cytoplasmic staining?
Expect a predominantly granular cytoplasmic pattern in tissue sections (HPA tissue IHC). CHCHD2 is mainly in the mitochondrial intermembrane space, while a nuclear location is also annotated (UniProt Q9Y6H1 subcellular location). Therefore, assess nuclear staining separately from cytoplasmic granules and record whether each pattern occurs in intact cells rather than damaged areas (UniProt Q9Y6H1; standard IHC practice). A predominantly diffuse nuclear signal deserves comparison with no-primary staining and a second validated readout before being assigned to CHCHD2 (standard IHC practice). HPA supports mitochondrial localisation in ICC/IF images, but its tissue IHC description alone does not establish nuclear staining specificity (HPA subcellular; HPA tissue IHC).
How do sequence features affect interpretation of unexpected staining?
The supplied record lists a 151-residue CHCHD2 chain, a CHCH domain at residues 111–151, and no annotated isoforms (UniProt Q9Y6H1). It also lists no transmembrane segment, signal peptide, glycosylation site or modified residue (UniProt Q9Y6H1 topology and processing). The antibody epitope is unspecified, so these annotations cannot identify which part of CHCHD2 the stain detects (datasheet A06266-1; UniProt Q9Y6H1). For discordant patterns, compare matched sections with an independently characterised antibody or an orthogonal expression readout, if available (standard IHC practice). Keep conclusions about isoform-specific staining or epitope masking provisional until the epitope is mapped (standard IHC practice).
How can I adapt this IHC result to a multiplex IF experiment?
Treat IF/ICC as a separate optimisation: the 1:200 overnight incubation and EDTA at pH 8.0 come from paraffin-section chromogenic IHC (datasheet A06266-1). HPA supports mitochondrial localisation in ICC/IF images, and UniProt places CHCHD2 mainly in the mitochondrial intermembrane space (HPA subcellular; UniProt Q9Y6H1). Choose permeabilisation that gives antibody access to that compartment, then check whether mitochondrial structure remains interpretable (UniProt Q9Y6H1; standard IF practice). Multiplex with a marker for the expected cell type and a mitochondrial marker where channel capacity permits (standard IF practice). Select spectrally separated fluorophores after measuring tissue autofluorescence, and include single-stain controls for bleed-through (standard IF practice).
How do I reduce diffuse brown staining without losing CHCHD2 signal?
Start by comparing the stained section with a no-primary control and inspect whether brown deposit follows cells, tissue edges or endogenous pigment (standard IHC practice). The selected protocol used 10% goat-serum blocking, 1:200 primary antibody overnight at 4°C, and peroxidase-based DAB detection (datasheet A06266-1). For persistent diffuse colour, optimise serum blocking, washing, primary concentration and DAB development on matched sections (standard IHC practice). Include a peroxidase block where appropriate for HRP detection and check its effect with controls (standard IHC practice). Preserve a granular cytoplasmic pattern during optimisation, rather than maximising total brown area (HPA tissue IHC).
What is a defensible way to quantify CHCHD2 IHC? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and viable tissue region before scoring, and exclude folds, necrosis and edge artefacts (standard IHC practice). Record the percentage of positive cells and staining intensity, then calculate an H-score from intensity categories if that scale suits the study (standard IHC practice). For granular staining, a positive-cell percentage or granule density per mm² can complement the H-score (HPA tissue IHC; standard IHC practice). Normalise area-based counts to viable tissue area and compare cell-based scores within the same cell type across specimens (standard IHC practice). Keep acquisition, thresholding and DAB development consistent because apparent intensity depends on the staining run (standard IHC practice).
When should I doubt an apparent CHCHD2-positive result?
A granular cytoplasmic pattern fits the tissue IHC profile, while mitochondrial localisation has independent ICC/IF support (HPA tissue IHC; HPA subcellular). Check whether the signal occurs in intact cells of the expected compartment and whether no-primary sections show similar deposit (UniProt Q9Y6H1 subcellular location; standard IHC practice). Edge-only staining, necrotic areas and residual endogenous peroxidase activity can create misleading DAB colour (standard IHC practice). Cell identity also matters: HPA reports high staining in several glandular and neuronal cell populations, but no detection in smooth muscle cells (HPA tissue IHC). Interpret discordant patterns cautiously because HPA rates tissue staining as Approved with medium agreement between staining and RNA expression (HPA tissue IHC).
Boster reagents
Best CHCHD2 / Coiled-coil-helix-coiled-coil-helix domain-containing protein 2 IHC Antibodies
A06266-1 has an IHC image from a human paraffin section (A06266-1 IHC caption). IF/ICC is listed without an IF image, and Human and Rat reactivity is listed (catalog applications, IF images, reactivity).
Real IHC data
Anti-CHCHD2 Antibody ®
Cat # A06266-1
A06266-1 is shown in chromogenic IHC on a paraffin section of human lung cancer tissue (A06266-1 IHC caption). It is also listed for IF/ICC and as reactive with Human and Rat, but no IF image is supplied (catalog applications, reactivity, IF images).
Which to pick: Choose A06266-1 for human paraffin-section IHC: its image caption reports EDTA retrieval at pH 8.0, primary antibody at 1:200 overnight, and HRP/DAB detection; the fixative is unreported (A06266-1 IHC caption). For IF/ICC, the same rabbit polyclonal antibody is listed at 1:50, without a supplied IF image (catalog host, dilution, applications, IF images). For rat samples, A06266-1 lists Rat reactivity, while its supplied IHC image shows human tissue only (catalog reactivity; A06266-1 IHC caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9Y6H1 (CHCH2_HUMAN, Coiled-coil-helix-coiled-coil-helix domain-containing protein 2).
- Human Protein Atlas. CHCHD2 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. CHCHD2 subcellular location (ICC-IF): Localized to the mitochondria..
- Human Protein Atlas. CHCHD2 antibody validation summary (2 antibodies).
- Neuropathologic description of CHCHD10 mutated amyotrophic lateral sclerosis. Neurology. Genetics 2020 — PMC6975173.
- Knockdown of CHCHD2 inhibits migration and angiogenesis of human renal cell carcinoma: A potential molecular marker for treatment of RCC. Oncology letters 2019 — PMC6313053.
- CHCHD2 is a potential prognostic factor for NSCLC and is associated with HIF-1a expression. BMC pulmonary medicine 2020 — PMC7020603.
- Decoding temporal heterogeneity in NSCLC through machine learning and prognostic model construction. World journal of surgical oncology 2024 — PMC11170806.
- PubMed PMID:12853948 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:21269460 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CHCHD2 (Coiled-coil-helix-coiled-coil-helix domain-containing protein 2). Expected localisation Observed granular cytoplasm (HPA tissue IHC); mainly mitochondrial intermembrane space (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A06266-1). Validated IHC antibody: A06266-1 at 1:50 recommended; image 1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Smooth muscle. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet A06266-1); 2. Fixation: Image fixative and duration unreported (datasheet A06266-1); 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 retrieval: EDTA pH 8.0 (datasheet A06266-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A06266-1); 8. Primary antibody: Rabbit anti-CHCHD2, 1:50 recommended; image 1:200 (datasheet A06266-1); 9. Primary incubation: Overnight at 4 °C (datasheet A06266-1). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: 0 isoforms annotated; intact 1–151 chain (UniProt); Regulation: Expression regulation is not annotated (UniProt). Evidence: UniProt Q9Y6H1, HPA tissue IHC · Approved, HPA ICC-IF · mitochondria., Datasheet A06266-1, PMC6313053. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CHCHD2/immunohistochemistry-ihc-guide.