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CNOT4 / CCR4-NOT transcription complex subunit 4 · IHC design guide
Design Immunohistochemistry for CNOT4
Plan paraffin-section IHC for CNOT4 using its widespread cytoplasmic tissue profile (HPA tissue IHC) and the catalog antibody’s 1:100–1:300 IHC dilution (datasheet). Compare cell types within the section, with adipocytes as a lower-staining reference (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CNOT4 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 (HPA tissue IHC); nuclear location also annotated (UniProt) | |
| Staining pattern | Widespread cytoplasmic staining across tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Low tissue RNA specificity (HPA tissue RNA) | |
| Isoform / epitope | 10 isoforms; epitope coverage needs checking (UniProt) |
Section 1
Recommended CNOT4 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is presented alongside one published CNOT4 IHC protocol from a non-small cell lung cancer model (PMC7714061).
| Sample | Paraffin-embedded human testis tissue; fixative not specified (datasheet A08405) |
| Fixation | Image fixative and duration unreported (datasheet A08405); 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 Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear 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-CNOT4, 1:100 - 1:300 (datasheet A08405) |
| 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 | CNOT4-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with pH 9.0 Tris-EDTA retrieval at 95–98 °C for 20 min for this nuclear antigen (page retrieval rule; UniProt O95628 localisation).
Section 2
What Is the Expected CNOT4 Staining Pattern?
In paraffin sections, expect predominantly cytoplasmic CNOT4 staining across many cell types, with strong staining reported in selected glandular, respiratory epithelial, neuronal and glial cells (HPA: ubiquitous cytoplasmic expression; High in listed cells). Nuclear localisation is also possible (UniProt O95628: cytoplasm and nucleus). CNOT4 has no transmembrane segment, so a membrane-only pattern is unexpected (UniProt O95628: topology). HPA rates its tissue IHC evidence Approved, with medium consistency against RNA data (HPA: reliability).
| Clear cytoplasmic staining in colon glandular cells, with an interpretable counterstain. | This matches a reported high-staining cell population (HPA: High in colon glandular cells) and the broad cytoplasmic tissue profile (HPA: ubiquitous cytoplasmic expression). Judge the pattern in its tissue context rather than requiring every cell to have equal intensity (HPA: Low in adipocytes). |
| Some nuclear signal accompanies cytoplasmic staining in otherwise plausible cells. | Nuclear localisation is biologically plausible (UniProt O95628: nucleus and cytoplasm), while the supplied tissue IHC summary describes cytoplasmic expression (HPA: tissue profile). Record nuclear and cytoplasmic scores separately; the HPA tissue summary does not establish the frequency or intensity of nuclear IHC staining. |
| A sharply membrane-bound or extracellular deposit dominates, without convincing cytoplasmic signal. | Treat this as a suspect compartment pattern: CNOT4 is assigned to cytoplasm and nucleus and has no transmembrane segment (UniProt O95628: subcellular location; topology). Inspect morphology and detection controls before attributing the deposit to CNOT4 (general IHC practice). |
| Strong staining appears chiefly in adipocytes while expected high-staining cells are faint. | That distribution conflicts with the supplied tissue observations (HPA: Low in adipocytes; High in colon glandular cells). Consider cross-reactivity or endogenous detection activity, then compare primary-omission and tissue controls (general IHC practice). Low HPA staining is not a claim that adipocytes must be negative. |
| Broad, hazy chromogen obscures cell boundaries, or a reported high-staining tissue has no signal. | Haze is difficult to score as cellular CNOT4 staining (general IHC practice). An absent signal in colon glandular cells or bronchial respiratory epithelial cells conflicts with reported high staining (HPA: High in those cells); check controls and assay performance before calling the specimen negative. |
💡Expected CNOT4 appearanceCall a slide positive when defined cytoplasmic staining is visible in an appropriate cell population, such as strongly stained colon glandular cells (HPA: High); nuclear signal may coexist (UniProt O95628: nucleus), whereas isolated membrane-like deposits are suspect (UniProt O95628: topology).
| Which tissue anchors interpretation? | Colon glandular cells and bronchial respiratory epithelial cells are reported High, while adipocytes are Low (HPA: tissue IHC). Use a reported high-staining population as a positive comparator; the supplied HPA record lists no negative tissue. |
| How strong is the antibody evidence? | The supplied antibody HPA005737 is IHC Approved (HPA: antibody validation), and the tissue profile has medium consistency with RNA data (HPA: reliability). These labels support comparison with the reported pattern; they do not establish every observed deposit as specific. |
| Could isoforms affect interpretation? | UniProt lists 10 CNOT4 isoforms (UniProt O95628: isoforms). The supplied record gives no antibody epitope or isoform coverage, so do not assign staining differences to a particular isoform without separate validation. |
| Does antigen retrieval have a CNOT4-specific setting? | No retrieval condition or target-specific fixation sensitivity is supplied (UniProt O95628 and HPA: supplied records). Retrieval is an IHC workflow variable; evaluate it against tissue controls without claiming a known CNOT4-specific effect (general IHC practice). |
| What should IF/ICC show? | Cytoplasmic or nuclear localisation is plausible (UniProt O95628: subcellular location). HPA provides no main ICC-IF location or cell-line images here (HPA: subcellular record), and the listed antibody has no supplied ICC validation (HPA: antibody record). This does not establish a validated IF/ICC staining pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| No chromogen in a reported high-staining cell population. | The run or detection system may have failed; the observation conflicts with the reported high tissue staining (HPA: High in colon glandular cells). | Check a same-run positive tissue, reagent application and detection controls before scoring the specimen as CNOT4 negative (general IHC practice). |
| Diffuse colour covers cells and stroma. | Nonspecific background or endogenous detection activity can obscure localisation (general IHC practice). | Review primary-omission controls, blocking and detection steps; score only discernible cellular staining (general IHC practice). |
| Staining is restricted to a cell edge or extracellular material. | That pattern conflicts with the assigned cytoplasmic and nuclear locations and absent transmembrane segment (UniProt O95628: location; topology). | Inspect morphology and controls, and seek a reproducible intracellular pattern before interpreting the deposit as CNOT4 (general IHC practice). |
| Adipocytes stain more strongly than the expected comparator. | Strong adipocyte staining is discordant with the supplied low-staining observation (HPA: Low in adipocytes); artefact or cross-reactivity is possible. | Compare a reported high-staining cell population and primary-omission control, then assess whether the pattern persists across sections (HPA: High in colon glandular cells; general IHC practice). |
| Nuclear staining appears without clear cytoplasmic staining. | Nuclear localisation is plausible (UniProt O95628: nucleus), but the supplied tissue IHC profile emphasises cytoplasm (HPA: tissue profile). | Score the compartments separately and check a reported high-staining tissue and controls; avoid treating nuclear-only IHC as an established HPA pattern. |
| Adjacent sections yield inconsistent apparent intensity. | Cell composition or uneven staining can change the field being compared (general IHC practice); HPA reports different levels across cell populations (HPA: High in colon glandular cells; Low in adipocytes). | Compare the same identified cell type in intact areas with matched counterstaining and same-run controls before concluding that CNOT4 expression changed (general IHC practice). |
Experiment evidence
IHC & IF images showing CNOT4 staining
A08405 · IHCImmunohistochemistry analysis of paraffin-embedded human testis tissue, using CNOT4 Antibody. The picture on the right is blocked with the s
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000080802-CNOT4/tissue/cerebral+cortex#imid_2269548
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000080802-CNOT4/tissue/colon#imid_2288902
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000080802-CNOT4/tissue/liver#imid_2288903
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000080802-CNOT4/tissue/kidney#imid_2288912
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CNOT4 IHC & IF
🧪Run adrenal gland first and look for CNOT4 staining in glandular cells (HPA: High in adrenal gland glandular cells). HPA detects CNOT4 in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and expect any cells without specific signal on the positive slide to show counterstain and background only, without assigning an unvalidated internal-negative cell type (HPA: no negative tissue rows; standard IHC practice).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: CNOT4 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for CNOT4; derive a cell-line control from the positive tissue's cell type (Glandular cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control and a control matched to the primary antibody’s host species and clonality, using matched isotype IgG for a monoclonal antibody or matched host IgG for a polyclonal antibody; add a CNOT4 knockout specimen if available or a peptide-block control, as shown for A08405 in human testis (standard IHC practice; A08405 tissue-IHC caption: peptide block). Check endogenous peroxidase background on the adrenal section during chromogenic detection (standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence; the A08405 paraffin-section testis caption does not state its fixative (A08405 tissue-IHC caption). Antigen-retrieval dependence is unreported, so assess retrieval conditions with the positive and negative controls (supplied evidence; standard IHC practice). The evidence does not establish that frozen sections or IF are easier for CNOT4, and no CNOT4-specific adrenal artefact is reported; monitor nonspecific peroxidase signal during chromogenic IHC (HPA subcellular: no ICC-IF image lines; supplied evidence; standard IHC practice).
HPA tissue IHC evidence for CNOT4
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 → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in molecular layer | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: CNOT4 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced CNOT4 IHC Tips
Troubleshoot CNOT4 staining in paraffin section IHC using its reported cytoplasmic and nuclear localisation, tissue staining profile, and the catalog antibody’s image controls.
What retrieval should I try first when CNOT4 staining is weak in paraffin sections?
Start with Tris-EDTA at pH 9.0, heated to 95–98 °C for 20 min (page retrieval setting). Keep section thickness, cooling, and detection conditions constant while comparing retrieved sections with a no-retrieval section, so a change in signal can be attributed to retrieval (standard IHC practice). Score cytoplasmic and nuclear staining separately because both locations are reported for CNOT4 (UniProt O95628: subcellular location). If the first run remains weak, test a shorter heating interval before increasing retrieval intensity, and inspect tissue morphology and negative controls for damage or nonspecific staining (standard IHC practice).
Could fixation explain weak CNOT4 staining in my paraffin sections?
Target-specific sensitivity of CNOT4 staining to fixation is unknown from the supplied evidence (selected A08405 tissue-IHC caption: fixative not stated). The catalog image shows staining in paraffin-embedded human testis tissue, but its caption does not identify the fixative or fixation duration (selected A08405 tissue-IHC caption). Record those variables for each specimen and compare sections processed together with the same retrieval and detection settings (standard IHC practice). If staining varies across specimens, inspect morphology and compare internal cell populations before attributing the difference to CNOT4 abundance; the reported cytoplasmic and nuclear locations alone cannot establish a fixation effect (UniProt O95628: subcellular location; standard IHC practice).
How should I troubleshoot CNOT4 staining that appears only in nuclei?
Assess nuclear and cytoplasmic staining as distinct readouts because CNOT4 is reported in both compartments (UniProt O95628: subcellular location). A nucleus-only pattern therefore warrants review of retrieval, counterstain intensity, and whether cytoplasmic signal remains visible in nearby well-preserved cells (standard IHC practice). Compare the distribution with the broadly cytoplasmic tissue profile, while recognising that this profile has only medium consistency with RNA expression data (HPA tissue IHC: ubiquitous cytoplasmic expression; Approved reliability description). Use a no-primary control to assess detection artefact, and avoid interpreting a compartment shift as a functional change without an independent assay (standard IHC practice).
Could isoforms or epitope masking explain uneven CNOT4 staining?
Check the antibody’s documented immunogen or epitope before assigning uneven staining to an isoform, because CNOT4 has 10 listed isoforms (UniProt O95628: isoforms). Map any known epitope against the 109–189 RNA recognition motif and recorded modified residues, including phosphoserines at 71, 301, 324, and 432 (UniProt O95628: domains and modified residues). Those features identify possible questions for epitope assessment; they do not establish masking or isoform specificity for this antibody (UniProt O95628: record; standard IHC interpretation). Compare staining across serial sections under matched retrieval conditions, and treat the catalog peptide-block image as a control for that tested staining pattern rather than proof of isoform selectivity (selected A08405 tissue-IHC caption; standard IHC practice).
How should I plan a CNOT4 immunofluorescence follow-up to my IHC result?
Use IHC to identify a candidate cell population, then multiplex CNOT4 with a validated marker for that population in the IF experiment (standard IF practice). For example, glandular cells in adrenal gland, appendix, and colon show high CNOT4 staining in the supplied tissue profile (HPA tissue IHC: listed positive cells). Choose spectrally separated fluorophores after checking tissue autofluorescence in an unstained section, and include single-label controls to assess channel bleed-through (standard IF practice). Because CNOT4 has no transmembrane segment and is reported in cytoplasm and nucleus, optimise permeabilisation for access to intracellular epitopes; the supplied HPA subcellular payload provides no ICC/IF images for direct comparison (UniProt O95628: topology and location; HPA subcellular: ICC/IF images).
What should I check when CNOT4 chromogenic staining is diffuse or dirty?
First compare the stained section with a no-primary control and inspect whether colour follows tissue edges, damaged regions, or the expected cellular compartments (standard IHC practice; UniProt O95628: cytoplasmic and nuclear location). Check the peroxidase-block step and examine a detection-only control if diffuse DAB signal persists; these are general chromogenic workflow checks, not CNOT4-specific findings (standard IHC practice). Titrate the primary antibody and detection conditions using matched sections, then compare cellular signal with surrounding background under the same imaging settings (standard IHC practice). Peptide blocking is shown for the catalog antibody in paraffin-embedded human testis tissue, but that comparison does not diagnose every source of background in another specimen (selected A08405 tissue-IHC caption).
How can I quantify CNOT4 IHC without confusing staining with cell composition? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and cytoplasmic or nuclear compartment before scoring, because CNOT4 is reported in both locations (UniProt O95628: subcellular location). Within each predefined region, record the percentage of positive cells and intensity categories to calculate an H-score from 0–300, or report positive-cell density per mm² when cell counts are the main outcome (standard IHC scoring practice). Normalise positive counts to all evaluable cells of the same type, or to the measured viable tissue area for density, while excluding damaged areas by a preset rule (standard IHC scoring practice). Keep retrieval, detection, thresholds, and imaging settings consistent across specimens, and report nuclear and cytoplasmic scores separately (standard IHC scoring practice; UniProt O95628: subcellular location).
How do I distinguish convincing CNOT4 staining from an IHC artefact?
Look for cellular staining that is reproducible across intact regions and assess cytoplasm and nucleus separately, since both are reported CNOT4 locations (UniProt O95628: subcellular location; standard IHC practice). Strong signal confined to section edges, necrotic tissue, or a no-primary control points to an artefact requiring review of staining and detection conditions (standard IHC practice). Check endogenous peroxidase contribution when DAB colour appears without primary antibody, and compare suspicious cells with a counterstained section to confirm their identity (standard chromogenic IHC practice). High staining in several listed glandular and neural cell populations and low staining in adipocytes provide context, but the HPA profile has medium consistency with RNA expression and cannot alone establish specificity in a new specimen (HPA tissue IHC: positive and low cells; reliability description).
Boster reagents
Best CNOT4 / CCR4-NOT transcription complex subunit 4 IHC Antibodies
A08405 has real IHC data from paraffin-embedded human testis (catalog IHC image caption); IF/ICC is listed without an IF figure (catalog applications and image captions). Human, mouse and rat reactivity is listed (catalog reactivity).
Real IHC data
Anti-CNOT4 Antibody
Cat # A08405
A08405 is the only card and shows IHC staining of paraffin-embedded human testis with a peptide-blocked comparison (catalog IHC image caption). IF and ICC are listed applications, and human, mouse and rat are listed as reactive species; no IF image is supplied (catalog applications, reactivity and image captions).
Which to pick: Choose A08405 for paraffin-section IHC because its own image shows human testis staining; the fixative is unreported (catalog IHC image caption). For IF/ICC, A08405 is listed at 1:50 for IF, but has no IF figure (catalog applications, IF dilution and image captions). For mouse or rat samples, A08405 lists reactivity with both species, though the supplied IHC image shows human tissue (catalog reactivity and IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O95628 (CNOT4_HUMAN, CCR4-NOT transcription complex subunit 4).
- Human Protein Atlas. CNOT4 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. CNOT4 subcellular location (ICC-IF): Highest expression in PODO/TERT256: 22.1 nTPM.
- Human Protein Atlas. CNOT4 antibody validation summary (1 antibodies).
- Aberrant expression of MYD88 via RNA-controlling CNOT4 and EXOSC3 in colonic mucosa impacts generation of colonic cancer. Cancer science 2021 — PMC8645755.
- CNOT4 enhances the efficacy of anti-PD-1 immunotherapy in a model of non-small cell lung cancer. FEBS open bio 2020 — PMC7714061.
- PubMed PMID:10637334 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:12853948 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CNOT4 (CCR4-NOT transcription complex subunit 4). Expected localisation Cytoplasmic staining (HPA tissue IHC); nuclear location also annotated (UniProt). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A08405 at 1:100 - 1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human testis tissue; fixative not specified (datasheet A08405); 2. Fixation: Image fixative and duration unreported (datasheet A08405); 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 Tris-EDTA buffer, pH 9.0 , 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-CNOT4, 1:100 - 1:300 (datasheet A08405); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: 10 isoforms ; epitope coverage needs checking (UniProt); Regulation: Low tissue RNA specificity (HPA tissue RNA). Evidence: UniProt O95628, HPA tissue IHC · Approved, HPA cell-line RNA · Highest expression in PODO/TERT256: 22.1 nTPM, Datasheet A08405, PMC7714061. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CNOT4/immunohistochemistry-ihc-guide.