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CLCF1 / Cardiotrophin-like cytokine factor 1 · IHC design guide
Design Immunohistochemistry for CLCF1
Plan CLCF1 chromogenic IHC in paraffin sections with bone marrow hematopoietic cells as a medium-staining reference and caudate glia as an undetected comparator (HPA tissue IHC). Start the catalog antibody at 1:50 (datasheet), and interpret cytoplasmic staining cautiously because this secreted protein’s tissue location may differ from its RNA location (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CLCF1 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); secreted protein (UniProt) | |
| Staining pattern | Cytoplasmic signal in several tissues; plasma positivity (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Bone marrow+4 more · see all | |
| Negative control | Caudate+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation and processing consistent across paraffin sections. (selected-SKU IHC image A08886) | |
| Caveat | RNA and protein locations may differ for this secreted protein (HPA tissue IHC) | |
| Regulation | No expression regulator specified (UniProt) | |
| Isoform / epitope | 2 isoforms; mature chain starts at 28; check epitope coverage (UniProt) |
Section 1
Recommended CLCF1 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet) with published CLCF1 chromogenic IHC protocols (PMC9073803; PMC12305993).
| Sample | Paraffin-embedded human brain tissue; fixative not specified (datasheet A08886) |
| Fixation | Image fixative and duration unreported (datasheet A08886); 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-CLCF1, 1:50 recommended; image 1:200 (datasheet A08886) |
| 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 | CLCF1-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in several tissues and positivity in plasma. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page antigen retrieval); adjust to the published workflow for the chosen sample.
Section 2
What Is the Expected CLCF1 Staining Pattern?
CLCF1 is secreted and has no transmembrane segment (UniProt Q9UBD9 topology). In tissue IHC, expect cytoplasmic staining in selected cells and positivity in plasma (HPA tissue IHC). HPA reports medium staining in bone marrow hematopoietic cells, lung macrophages and several other cell populations, but rates the tissue pattern Uncertain because a secreted protein’s tissue RNA and protein locations may differ (HPA tissue IHC).
| Cytoplasmic staining in bone marrow hematopoietic cells or lung macrophages. | This fits reported medium staining in those cells (HPA tissue IHC). Score the stained cells and their intensity; nearby unstained cells do not negate a cell-specific result (general IHC practice). |
| Predominantly nuclear or sharply membrane-bound signal in tissue sections. | That differs from HPA’s cytoplasmic tissue profile and merits review for artefact (HPA tissue IHC). Nuclear bodies are reported separately by ICC-IF; that result does not establish a nuclear tissue-IHC pattern (HPA ICC-IF). |
| Strong staining in a cell population HPA lists as not detected, such as prostate glandular cells. | Treat this as discordant with the reference pattern, not proof of CLCF1 expression (HPA tissue IHC). Check for antibody cross-reactivity or endogenous chromogen activity (general IHC practice). |
| Diffuse chromogen across cells and surrounding section, with little separation between them. | This is difficult to score as cell-specific staining (general IHC practice). HPA reports cytoplasmic expression and plasma positivity, so extracellular signal alone cannot identify its cellular source (HPA tissue IHC). |
| No signal in a section of bone marrow that contains hematopoietic cells. | HPA reports medium staining in those cells, so review the assay and tissue before interpreting the absence (HPA tissue IHC). The HPA tissue result is rated Uncertain and is not a guaranteed positive control. |
💡Expected CLCF1 appearanceA plausible positive is medium cytoplasmic staining in HPA-listed cells, such as bone marrow hematopoietic cells or lung macrophages; strong staining confined to an HPA-listed negative cell population warrants scrutiny (HPA tissue IHC).
| Secreted protein and topology (UniProt Q9UBD9). | CLCF1 lacks a transmembrane segment and its secretion depends on CRLF1 or soluble CNTFR (UniProt Q9UBD9); do not require a crisp membrane outline when judging tissue IHC. |
| Mature protein processing (UniProt Q9UBD9). | The precursor has a signal peptide at residues 1–27; the annotated mature chain spans 28–225 (UniProt Q9UBD9). No antibody epitope is supplied, so epitope-specific staining predictions are unsupported. |
| Observed positive cell populations (HPA tissue IHC). | HPA reports medium staining in breast myoepithelial cells, bronchial epithelium, cardiomyocytes, kidney tubules, skeletal myocytes and skin Langerhans cells (HPA tissue IHC). |
| Reference negatives and low staining (HPA tissue IHC). | HPA lists prostate and salivary glandular cells as not detected, while cerebral cortex and hippocampal neurons are low (HPA tissue IHC). A low category is not a strict negative. |
| Confidence in tissue interpretation (HPA tissue IHC). | HPA rates tissue IHC Uncertain and notes pending external verification; HPA042444 is also rated Uncertain for IHC (HPA antibodies). Use the pattern as a reference, not definitive identity. |
| IF/ICC Q: Should nuclear bodies or vesicles define the IHC positive pattern? | A: No. HPA approves nuclear-body and vesicle localisation in ICC-IF (HPA ICC-IF), while its tissue-IHC profile is cytoplasmic with plasma positivity (HPA tissue IHC). Evaluate each application against its own evidence. |
| Situation | Likely cause | Next action |
|---|---|---|
| Bone marrow hematopoietic cells show no detectable chromogen. | Weak assay detection or an unsuitable section is possible (general IHC practice); HPA’s medium result does not establish a required outcome (HPA tissue IHC). | Confirm that the expected cells are present, inspect the counterstain and detection controls, and review the antibody’s IHC-P conditions (general IHC practice). |
| The section is uniformly brown, including HPA-listed negative cells. | Non-specific binding, residual endogenous peroxidase activity or excess chromogen can obscure cell-specific staining (general IHC practice). | Review blocking and detection controls, compare HPA-listed negative cells, and assess whether staining remains cytoplasmic in listed positives (general IHC practice; HPA tissue IHC). |
| Prostate or salivary glandular cells stain strongly. | The result conflicts with HPA’s not-detected calls; cross-reactivity or endogenous detection activity is possible (HPA tissue IHC; general IHC practice). | Check an appropriate negative detection control and compare morphology and staining distribution before assigning CLCF1 positivity (general IHC practice). |
| Only nuclei appear positive in chromogenic tissue IHC. | This does not match HPA’s cytoplasmic tissue profile, although nuclear bodies are approved in ICC-IF (HPA tissue IHC; HPA ICC-IF). | Review counterstain and chromogen localisation under brightfield, then score the tissue-IHC result against the tissue reference (general IHC practice; HPA tissue IHC). |
| Staining appears mainly in plasma or extracellular spaces. | HPA reports plasma positivity, and UniProt annotates CLCF1 as secreted (HPA tissue IHC; UniProt Q9UBD9). This distribution alone does not identify a producing cell. | Record extracellular and cellular staining separately; use the HPA-listed cell populations when judging whether the cellular pattern agrees (general IHC practice; HPA tissue IHC). |
| ICC-IF shows puncta, but the tissue IHC section lacks punctate staining. | HPA reports approved vesicle and nuclear-body localisation for ICC-IF, while tissue IHC is described as cytoplasmic (HPA ICC-IF; HPA tissue IHC). | Interpret the assays separately. For the IHC section, document cell type, cytoplasmic distribution and intensity against the tissue profile (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing CLCF1 staining
A08886 · IHCImmunohistochemical analysis of paraffin-embedded human-brain, antibody was diluted at 1:200
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000175505-CLCF1/tissue/cerebral+cortex#imid_11690030
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000175505-CLCF1/tissue/colon#imid_11688220
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000175505-CLCF1/tissue/liver#imid_11688237
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000175505-CLCF1/tissue/kidney#imid_11688238
HPA · ICC-IF SiHaHuman Protein Atlas immunofluorescence, SiHa, antibody HPA042444; green = CLCF1, blue = nucleus, red = microtubules.
HPA · ICC-IF SiHaHuman Protein Atlas immunofluorescence, SiHa, antibody HPA042444; green = CLCF1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CLCF1 IHC & IF
🧪Run bone marrow first and assess staining in hematopoietic cells (HPA: bone marrow hematopoietic cells, Medium). Use caudate glial cells as the negative tissue comparison (HPA: caudate glial cells, Not detected); cells on the positive slide without specific chromogen deposition should show background only, although HPA does not identify a validated internal-negative cell population (HPA: bone marrow hematopoietic cells, Medium).
Positive control tissue: Bone marrow (Hematopoietic cells, HPA Medium)
Negative control tissue: Caudate (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CLCF1 in SiHa, U-251MG, U2OS, with annotated localisation: Nuclear bodies (approved), Vesicles (approved) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, an isotype control matched to the primary antibody’s host species and immunoglobulin class, and a validated CLCF1 knockout specimen or cognate peptide block if available (standard IHC practice). Quench endogenous peroxidase and check for residual background in bone marrow before interpreting chromogenic staining (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and retrieval dependency are unreported in the supplied evidence; the catalog antibody’s paraffin-section brain caption gives a 1:200 dilution but does not report the fixative (A08886 caption: paraffin-embedded human brain, 1:200; fixative not stated). The supplied evidence does not establish whether frozen sections or IF are easier for CLCF1, although ICC-IF images exist for SiHa, U-251MG and U2OS (HPA: CLCF1 subcellular record). In bone marrow, endogenous peroxidase can complicate chromogenic interpretation, so assess the quenched no-primary control alongside the stained section (standard IHC practice).
HPA tissue IHC evidence for CLCF1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Secreted protein, tissue location of RNA and protein is expected to differ. Pending external verification.). 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 |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Breast | Myoepithelial cells | Medium | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Medium | Protein (IHC) | HPA → |
| Kidney | Cells in tubules | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Prostate | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Salivary gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CLCF1 IHC Tips
Troubleshoot CLCF1 staining in paraffin sections with chromogenic IHC, using the catalog image and reported tissue patterns as starting points.
How should I adjust retrieval when CLCF1 staining is weak in paraffin sections?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval setting). Let sections cool in the retrieval buffer before washing, and compare a no-primary control with the 1:200 catalog antibody dilution shown in paraffin-embedded human brain (standard IHC practice; selected-SKU A08886 caption). If staining remains weak, test a longer retrieval interval on adjacent sections while checking whether tissue morphology deteriorates (standard IHC practice). CLCF1 has a cleaved signal peptide and no transmembrane segment, so staining need not outline the plasma membrane (UniProt Q9UBD9 topology and processing).
Could fixation explain variable CLCF1 staining across my paraffin sections?
The selected-SKU paraffin-section caption does not report its fixative, and target-specific CLCF1 sensitivity to fixation is unknown (selected-SKU A08886 caption). Record each specimen’s fixative and fixation duration, then compare adjacent sections using the same citrate pH 6.0, 95–98 °C, 20 min retrieval and detection run (page retrieval setting; standard IHC practice). Keep section thickness, antibody incubation, and the catalog image’s 1:200 starting dilution consistent during that comparison (selected-SKU A08886 caption; standard IHC practice). If signal varies, assess morphology and processing records before attributing the difference to CLCF1 abundance (standard IHC practice).
Where should I expect CLCF1 signal, and is membrane staining credible?
Assess cytoplasmic staining and material outside cells separately: tissue IHC reports cytoplasmic expression in several tissues and positivity in plasma, although its reliability is uncertain (HPA tissue IHC). CLCF1 is secreted, has no transmembrane segment, and can associate with CRLF1 or soluble CNTFR, so a continuous membrane outline is not its expected topology (UniProt Q9UBD9 subcellular location and topology). Compare the pattern at the catalog image’s 1:200 dilution against no-primary and adjacent-section controls (selected-SKU A08886 caption; standard IHC practice). Nuclear bodies and vesicles are approved ICC/IF locations, but they do not establish a nuclear IHC scoring rule (HPA subcellular).
How can epitope placement affect interpretation of CLCF1 staining?
Confirm the antibody’s immunogen or epitope before using a negative section to exclude CLCF1, because the supplied caption gives a 1:200 dilution but no epitope (selected-SKU A08886 caption). The precursor has 225 aa, a signal peptide at residues 1–27, a mature chain at 28–225, and a glycosylation site at residue 29 (UniProt Q9UBD9 processing and glycosylation). Two isoforms are annotated, but their sequence differences and antibody coverage are not supplied here (UniProt Q9UBD9 isoforms; selected-SKU A08886 caption). Compare staining after the page’s citrate pH 6.0 retrieval, and interpret discordance only after checking documented epitope coverage (page retrieval setting; standard IHC practice).
How should I adapt the CLCF1 question to multiplex IF?
For multiplex IF, pair CLCF1 with a macrophage marker when examining lung, where macrophages show medium tissue-IHC staining (HPA tissue IHC; standard IF practice). Choose a far-red fluorophore for the weaker channel and inspect an unstained section to gauge tissue autofluorescence before setting exposure (standard IF practice). For intracellular vesicle or nuclear-body imaging, a brief 0.1% detergent permeabilisation is a starting condition; omit permeabilisation when testing an accessible extracellular epitope, and verify which pool the antibody recognizes (HPA subcellular; UniProt Q9UBD9 topology; standard IF practice). The 1:200 paraffin IHC caption does not establish an IF dilution or IF fixation condition (selected-SKU A08886 caption).
What should I check when CLCF1 chromogenic staining looks diffuse?
Run no-primary and secondary-only controls, and compare them with a section stained at the catalog image’s 1:200 dilution (selected-SKU A08886 caption; standard IHC practice). Include a peroxide block before peroxidase detection, then inspect DAB deposition in both controls; these are general chromogenic workflow checks, not CLCF1-specific validation (standard IHC practice). Reduce primary concentration or incubation time only after confirming that retrieval at citrate pH 6.0, 95–98 °C for 20 min preserves morphology (page retrieval setting; standard IHC practice). Diffuse extracellular signal requires cautious interpretation because CLCF1 is secreted and tissue location may differ from the site of expression (UniProt Q9UBD9 subcellular location; HPA reliability description).
How should I score CLCF1 across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored cell population and compartment before analysis, then report the percentage of positive cells and an H-score from intensity categories 0–3 (standard IHC practice). For dispersed positive cells, also report positive-cell density per mm² of viable tissue and normalize counts to the area and cell population actually evaluated (standard IHC practice). Use the same citrate pH 6.0 retrieval and detection settings across compared sections, with the catalog image’s 1:200 dilution as a documented starting point (page retrieval setting; selected-SKU A08886 caption; standard IHC practice). Keep extracellular deposits separate from cellular scores because CLCF1 is secreted and HPA tissue-IHC reliability is uncertain (UniProt Q9UBD9 subcellular location; HPA tissue IHC).
How do I distinguish convincing CLCF1 signal from artefact?
Look for a reproducible cell-associated pattern in the expected population: lung macrophages and bone-marrow hematopoietic cells have medium reported staining, while caudate glial cells were not detected (HPA tissue IHC). Treat a sharp tissue-edge rim, necrotic-area staining, or DAB present in no-primary controls as possible processing or endogenous-enzyme artefact, and check the peroxide block (standard IHC practice). A continuous plasma-membrane outline alone is weak evidence for CLCF1 because it has no transmembrane segment; extracellular staining remains plausible for a secreted protein (UniProt Q9UBD9 topology and subcellular location). Interpret even well-controlled staining cautiously because HPA labels its tissue-IHC reliability uncertain and notes that RNA and protein locations can differ (HPA tissue IHC).
Boster reagents
Best CLCF1 / Cardiotrophin-like cytokine factor 1 IHC Antibodies
A08886 has an IHC image of paraffin-embedded human brain (catalog image caption). IF is listed, with Human, Mouse, and Rat reactivity (catalog applications/reactivity); no IF image is supplied (catalog IF image alts).
Real IHC data
Anti-NNT-1 CLCF1 Antibody
Cat # A08886
A08886 has an IHC image of paraffin-embedded human brain stained at 1:200 (catalog image caption). It lists IHC and IF applications and Human, Mouse, and Rat reactivity (catalog applications/reactivity), but provides no IF image (catalog IF image alts).
Which to pick: For tissue IHC, choose A08886: its own image shows a paraffin-embedded human brain section at 1:200 (catalog image caption); the fixative is unreported (catalog image caption). For IF, A08886 lists a 1:50 dilution (catalog applications/IF dilution), while ICC validation and an IF image are unreported (catalog applications/IF image alts). For cross-species work, A08886 lists Human, Mouse, and Rat reactivity (catalog reactivity); its clone status is unreported (catalog clone).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9UBD9 (CLCF1_HUMAN, Cardiotrophin-like cytokine factor 1).
- Human Protein Atlas. CLCF1 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. CLCF1 subcellular location (ICC-IF): Localized to the nuclear bodies and vesicles..
- Human Protein Atlas. CLCF1 antibody validation summary (1 antibodies).
- CLCF1 is up-regulated in renal ischemia reperfusion injury and may associate with FOXO3. Annals of translational medicine 2022 — PMC9073803.
- CLCF1 Is a Novel Potential Immune-Related Target With Predictive Value for Prognosis and Immunotherapy Response in Glioma. Frontiers in immunology 2022 — PMC8898905.
- CLCF1 contributes to high-glucose-induced EndMT by modulating JAK2/STAT3 signaling. European journal of medical research 2025 — PMC12305993.
- Saikosaponin D and paeoniflorin improve the HCC immune microenvironment via CLCF1/PD-L1 mediated crosstalk between CAFs and tumor cells. Scientific reports 2026 — PMC13234363.
- PubMed PMID:10500198 — UniProt-cited evidence.
- PubMed PMID:10448081 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CLCF1 (Cardiotrophin-like cytokine factor 1). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC); secreted protein (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A08886 at 1:50 recommended; image 1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Bone marrow (Hematopoietic cells); negative Caudate. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human brain tissue; fixative not specified (datasheet A08886); 2. Fixation: Image fixative and duration unreported (datasheet A08886); 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-CLCF1, 1:50 recommended; image 1:200 (datasheet A08886); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation and processing consistent across paraffin sections. (selected-SKU IHC image A08886); Caveat: RNA and protein locations may differ for this secreted protein (HPA tissue IHC); Isoform / epitope: 2 isoforms; mature chain starts at 28 ; check epitope coverage (UniProt); Regulation: No expression regulator specified (UniProt). Evidence: UniProt Q9UBD9, HPA tissue IHC · Uncertain, HPA ICC-IF · nuclear bodies and vesicles., Datasheet A08886, PMC9073803, PMC12305993. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CLCF1/immunohistochemistry-ihc-guide.