CNTF / Ciliary neurotrophic factor · IHC design guide

Design Immunohistochemistry for CNTF

Plan chromogenic CNTF IHC on paraffin sections using caudate neurons and cerebellar Purkinje cells as positive references (HPA tissue IHC). Interpret staining with care because the precursor is probably cytoplasmic and CNTF may be released from damaged cells (UniProt).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for CNTF (IHC for CNTF): expected localisation Cytoplasmic precursor; secretion is possible (UniProt), antibody PB9012, validated IHC image, and IHC protocol steps — Printable **CNTF** IHC protocol sheet — expected localisation Cytoplasmic precursor; secretion is possible (UniProt), antibody PB9012, controls and protocol steps. Open the full CNTF IHC guide →

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CNTF 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 precursor; secretion is possible (UniProt)
	Staining pattern	Mainly CNS neurons, including Purkinje cells (HPA tissue IHC)
	Antigen retrieval	Citrate pH 6 HIER, heat-mediated (datasheet PB9012)
	Positive control ⓘ	Caudate+2 more · see all
	Negative control ⓘ	Adipose tissue+4 more · see all

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Keep fixation consistent across sections (standard IHC practice; not target-specific)
	Caveat	Presumed off-target binding may confound scoring (HPA tissue IHC)
	Regulation	No expression regulator annotated (UniProt)
	Isoform / epitope	No annotated isoforms or cleavage; epitope location unknown (UniProt)

Section 1

Recommended CNTF IHC & IF Protocols

The catalog antibody’s paraffin IHC protocol (datasheet: PB9012) is accompanied by published CNTF staining protocols for mouse hypothalamus (PMC3873503) and human prostate tissue (PMC7586252).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human intestinal cancer tissues; fixative not specified (datasheet PB9012)
Fixation	Image fixative and duration unreported (datasheet PB9012); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat retrieval: Citrate pH 6, 20 min (datasheet PB9012)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet PB9012)
Primary antibody	Rabbit anti-CNTF, 0.5-1μg/ml (datasheet PB9012)
Primary incubation	Overnight at 4 °C (datasheet PB9012)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9012)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	CNTF-positive staining in neuronal cells of caudate (HPA tissue IHC: High). HPA tissue profile: Expression mainly in CNS. No signal in the no-primary control.

💡Decision noteFor paraffin sections, start with heat-mediated citrate pH 6 retrieval (datasheet: PB9012; PMC7586252). The free-floating mouse protocol does not report CNTF retrieval (PMC3873503).

Section 2

What Is the Expected CNTF Staining Pattern?

CNTF is annotated as cytoplasmic and secreted, with no transmembrane segment (UniProt P26441). On paraffin IHC sections, look for staining in caudate neuronal cells and cerebellar Purkinje cells; cortical neuronal staining is weaker (HPA tissue IHC: High, High, and Medium, respectively). HPA rates the tissue pattern Approved and notes that presumed off-target binding was observed and disregarded (HPA tissue IHC: reliability description).

What am I looking at on my slide?
Caudate neurons or cerebellar Purkinje cells show prominent cellular staining.	This fits the reported High staining in those cell types (HPA tissue IHC). Assess which cells carry the signal, its intensity, and its compartment together; a dark field alone does not establish a specific CNTF result (general IHC practice).
Staining appears predominantly nuclear or outlines cell membranes without a discernible cellular component.	That distribution needs scrutiny: CNTF is annotated as cytoplasmic and secreted, with no transmembrane segment (UniProt P26441). HPA reports vesicles in ICC-IF, but that observation does not define a required paraffin IHC pattern (HPA subcellular ICC-IF).
Strong staining appears in adipocytes or bone-marrow hematopoietic cells.	Those sampled cell types were Not detected by HPA tissue IHC; the mismatch raises concern for cross-reactivity or endogenous detection activity (HPA tissue IHC; general IHC practice). Check the tissue-specific background and detection controls before assigning the signal to CNTF.
Color is widespread across cells and extracellular spaces, with little separation between expected positive cells and surrounding tissue.	The diffuse appearance is difficult to reconcile with HPA's mainly CNS tissue profile and its named positive cell types (HPA tissue IHC). Uneven blocking, excessive detection signal, or nonspecific binding are general IHC possibilities; control sections help distinguish them (general IHC practice).
Caudate neuronal cells and cerebellar Purkinje cells show no convincing signal.	These are reported High-staining cell types, so an absent result calls for a workflow check (HPA tissue IHC). Verify tissue identity and integrity, antibody and detection steps, and a known working positive section before interpreting another specimen as CNTF negative (general IHC practice).

💡Expected CNTF appearanceA convincing positive result shows prominent staining in caudate neuronal cells or cerebellar Purkinje cells, with less intense cortical neuronal staining; isolated nuclear, membrane-outline, or broadly diffuse staining warrants review (HPA tissue IHC: High/High/Medium; UniProt P26441: cytoplasmic, secreted, no transmembrane segment; general IHC practice).

How each factor affects the staining
Cell type and tissue selection	Caudate neurons and cerebellar Purkinje cells are High, while cortical neurons are Medium (HPA tissue IHC). Use the reported cell type, as well as the tissue, when judging a positive section (general IHC practice).
Compartment interpretation	CNTF is annotated as cytoplasmic and secreted and lacks a transmembrane segment (UniProt P26441). Vesicle localisation is an ICC-IF observation; it does not establish the exact appearance of chromogenic paraffin IHC (HPA subcellular ICC-IF).
Antibody evidence	The listed antibody HPA019654 has Approved IHC status; HPA's tissue summary also notes presumed off-target binding that was disregarded (HPA antibodies; HPA tissue IHC). Evaluate unexpected staining against controls rather than treating approval as proof of every stained structure (general IHC practice).
RNA versus protein readouts	Bone marrow is tissue enhanced at the RNA level, yet hematopoietic cells are Not detected in the supplied protein IHC record (HPA tissue IHC). An RNA designation alone does not predict a positive stain in that sampled cell type.
Molecular form	The record lists one CNTF chain, residues 1–200, with no annotated signal peptide, propeptide, isoforms, or glycosylation sites (UniProt P26441). It provides no basis for predicting a distinct processed-form or glycosylation-specific IHC pattern.
IF/ICC question: should staining be vesicular?	HPA reports approved vesicle localisation in ICC-IF images from A-431, U-251MG, and U2OS (HPA subcellular ICC-IF). Use that as IF context; the paraffin tissue IHC evidence separately identifies positive tissues and cell types (HPA tissue IHC).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
No staining in a reported High-positive cell type.	A failed or weak assay is possible when caudate neurons or Purkinje cells are blank (HPA tissue IHC; general IHC practice).	Confirm section identity and preservation, then review retrieval, antibody incubation, detection, and a working positive section without assuming a CNTF-specific fixation effect (general IHC practice).
All cells have a similar brown haze.	Nonspecific binding or excessive chromogenic development can obscure the expected cell pattern (general IHC practice; HPA tissue IHC: named positive cell types).	Compare reagent controls, inspect blocking and wash steps, and adjust detection development using the assay's validated procedure (general IHC practice).
Signal appears in cells reported Not detected.	Cross-reactivity or endogenous detection activity is possible in an unexpected cell type (HPA tissue IHC: adipocytes and bone-marrow hematopoietic cells Not detected; general IHC practice).	Check an appropriate no-primary control and the detection system's background controls; review the stained cell identity before reporting CNTF positivity (general IHC practice).
Only nuclei or cell borders stain strongly.	This is discordant with cytoplasmic/secreted annotation and absent transmembrane topology (UniProt P26441).	Inspect morphology and counterstain, compare positive and negative controls, and reassess whether the signal tracks the expected cell types (HPA tissue IHC; general IHC practice).
Cortex is weaker than caudate or cerebellum.	That difference matches HPA's Medium cortical neuronal level versus High caudate neuronal and Purkinje-cell levels (HPA tissue IHC).	Score each cell type against its reported level and the same run's controls; do not require equal intensity across these tissues (HPA tissue IHC; general IHC practice).
A vesicular IF image seems inconsistent with the IHC section.	HPA's vesicle assignment comes from ICC-IF, whereas the supplied paraffin IHC summary reports tissue and cell-type staining (HPA subcellular ICC-IF; HPA tissue IHC).	Interpret each assay against its own evidence; for IHC, prioritize the reported positive cells and control behavior (HPA tissue IHC; general IHC practice).

Source: UniProt P26441 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing CNTF staining

IHC analysis of CNTF using anti-CNTF antibody (PB9012). CNTF was detected in paraffin-embedded section of human intestinal cancer tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-CNTF Antibody (PB9012) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

PB9012 · IHCIHC analysis of CNTF using anti-CNTF antibody (PB9012). CNTF was detected in paraffin-embedded section of human intestinal cancer tissues. H

IHC analysis of CNTF using anti-CNTF antibody (PB9012). CNTF was detected in paraffin-embedded section of human lung cancer tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-CNTF Antibody (PB9012) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

PB9012 · IHCIHC analysis of CNTF using anti-CNTF antibody (PB9012). CNTF was detected in paraffin-embedded section of human lung cancer tissues. Heat me

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000242689-CNTF/tissue/cerebral+cortex#imid_20611875

HPA · CerebellumHuman Protein Atlas IHC, Cerebellum. Source: https://www.proteinatlas.org/ENSG00000242689-CNTF/tissue/cerebellum#imid_20611886

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000242689-CNTF/tissue/colon#imid_20611793

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000242689-CNTF/tissue/liver#imid_20611802

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA019654; green = CNTF, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for CNTF IHC & IF

🧪Run cerebellum first: Purkinje cells should stain High (HPA: cerebellum, Purkinje cells High). Use adipose tissue as the negative tissue because adipocytes are Not detected (HPA: adipose tissue, adipocytes Not detected); on the cerebellum slide, cells without specific CNTF signal should remain at background rather than be scored as positive.

Positive control tissue: Caudate (Neuronal cells, HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CNTF in A-431, U-251MG, U2OS, with annotated localisation: Vesicles (approved) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control and a matched rabbit IgG isotype control (selected-SKU caption: rabbit anti-CNTF primary); use CNTF-knockout material or validated immunizing-peptide competition as a biological negative (standard IHC practice). Check endogenous peroxidase and biotin background in the cerebellum when using the caption’s streptavidin–biotin and DAB detection (selected-SKU caption: SABC/DAB; standard IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU caption: fixative unreported). Citrate retrieval at pH 6 for 20 minutes was used in that IHC example, but whether CNTF staining depends on retrieval is unreported (selected-SKU caption: citrate retrieval); the supplied evidence does not establish that frozen sections or IF are easier. If adapting the assay to cerebellar IF, assess tissue autofluorescence before scoring signal (standard IF practice).

HPA tissue IHC evidence for CNTF

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Presumed off target binding observed and disregarded.). 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
Caudate	Neuronal cells	High	Protein (IHC)	HPA →
Cerebellum	Purkinje cells	High	Protein (IHC)	HPA →
Cerebral cortex	Neuronal 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 →
Breast	Adipocytes	Not detected	Protein (IHC)	HPA →

Section 3

Advanced CNTF IHC Tips

Use the catalog antibody’s paraffin section workflow as a starting point, then judge CNTF staining against compartment, cell type, and control tissue evidence (datasheet PB9012; UniProt P26441; HPA tissue IHC).

How should I adjust retrieval if CNTF staining is weak in paraffin sections?

Start with heat-mediated citrate retrieval at pH 6 for 20 minutes on paraffin sections (datasheet PB9012). Keep section thickness, heating, and cooling consistent across the test and control slides so staining differences can be attributed to the variable under study (standard IHC practice). If staining remains weak, compare a longer citrate retrieval with the original condition on adjacent sections, watching for tissue damage or increased background (standard IHC practice). Assess the result in neuronal cells from caudate or Purkinje cells from cerebellum, which have reported high staining, before changing the detection system (HPA tissue IHC).

Can fixation explain absent CNTF staining in a paraffin section?

CNTF-specific fixation sensitivity is unknown because the selected paraffin section caption does not state a fixative (datasheet PB9012). Record the fixative, time before fixation, and fixation duration for each specimen, then compare sections with matched processing and the same citrate retrieval at pH 6 (standard IHC practice; datasheet PB9012). Include a concurrently processed positive control, such as caudate neuronal cells or cerebellar Purkinje cells, to distinguish a failed staining run from a specimen-specific result (HPA tissue IHC; standard IHC practice). If the control stains and the specimen does not, report the specimen result with its processing history rather than assigning a CNTF-specific fixation effect (standard IHC practice).

Where should convincing CNTF staining appear in chromogenic IHC?

Evaluate cytoplasmic staining in the relevant cells, while allowing a vesicular pattern: CNTF is annotated as cytoplasmic or secreted, and vesicles are the approved subcellular location (UniProt P26441; HPA subcellular). CNTF has no annotated transmembrane segment, so a crisp membrane-only rim needs independent validation before it is called specific (UniProt P26441 topology; standard IHC practice). Use caudate neuronal cells and cerebellar Purkinje cells as anatomical reference patterns because both show high tissue staining (HPA tissue IHC). Document whether signal is intracellular, extracellular, or associated with damaged areas; the proposed release from damaged cells makes extracellular staining alone difficult to assign to the cell that produced CNTF (UniProt P26441).

How do unknown epitope details affect CNTF IHC interpretation?

Treat the recognized epitope as unmapped in this evidence set and avoid assigning staining to a particular CNTF region (datasheet PB9012). UniProt annotates one chain spanning residues 1–200, with no listed isoforms, signal peptide, propeptide, glycosylation sites, or modified residues (UniProt P26441). Those annotations do not establish whether fixation or citrate retrieval preserves this antibody’s binding site, so compare retrieval conditions using the same positive control and detection settings (UniProt P26441; standard IHC practice). If a second antibody is available, concordant staining with an independently mapped epitope can strengthen specificity, but record the epitope evidence rather than assuming the antibodies are independent (standard IHC practice).

How should I extend this IHC result to multiplex IF?

For a separate IF experiment, pair CNTF with a marker that identifies the neuronal or Purkinje cell population being evaluated; these populations show high tissue IHC staining (HPA tissue IHC; standard IF practice). Choose fluorophores after imaging unstained tissue, placing the weaker signal in a channel with less tissue autofluorescence and checking each channel for bleed-through (standard IF practice). CNTF is annotated as cytoplasmic and vesicular, with no transmembrane segment, so test permeabilisation when the antibody must reach an intracellular epitope; its precise epitope is unspecified here (UniProt P26441; HPA subcellular; datasheet PB9012). The catalog caption establishes a paraffin section chromogenic workflow, including citrate at pH 6, rather than an IF condition (datasheet PB9012).

What should I check when CNTF DAB staining looks diffuse?

First compare the stained section with a no-primary control and inspect whether diffuse brown signal follows tissue edges, damaged regions, or endogenous pigment (standard IHC practice). The selected workflow uses a biotinylated secondary, streptavidin-biotin complex, and DAB, so test for background from the detection reagents and include an appropriate endogenous peroxidase block (datasheet PB9012; standard IHC practice). The catalog incubation is 1 μg/ml overnight at 4°C after 10% goat serum blocking; vary antibody concentration only while holding retrieval and detection constant (datasheet PB9012; standard IHC practice). HPA notes presumed off-target binding was disregarded, so require convincing cell and compartment patterns before accepting widespread signal (HPA tissue IHC).

How can I score CNTF IHC across specimens reproducibly? ⚠ ANSWER MARKED FOR VERIFICATION

Define the anatomical compartment and eligible cell population before scoring, then use the same threshold and imaging settings for every section (standard IHC practice). Report the percentage of positive cells and staining intensity, or calculate an H-score from percentages at intensity grades 0–3; record whether extracellular signal was excluded (standard IHC practice; UniProt P26441). For spatial analyses, report positive cells per mm² of viable tissue and normalise to the area or eligible cell count, rather than total section area when tissue loss varies (standard IHC practice). Keep neuronal and Purkinje cell results separate from other populations because their reported staining levels provide distinct reference contexts (HPA tissue IHC).

How do I distinguish true CNTF staining from artefact?

A credible result has reproducible staining in an appropriate cell population and an intracellular pattern compatible with cytoplasmic or vesicular CNTF (HPA tissue IHC; UniProt P26441; HPA subcellular). Caudate neuronal cells and cerebellar Purkinje cells are reported high, whereas adipose adipocytes and adrenal glandular cells are reported undetected, making matched sections useful as contextual controls (HPA tissue IHC; standard IHC practice). Question isolated membrane rims, section-edge staining, necrotic deposits, or signal reproduced in a no-primary control; endogenous peroxidase can also produce misleading DAB color (UniProt P26441 topology; standard IHC practice). Interpret extracellular signal cautiously because CNTF may be released by damaged cells, and HPA explicitly notes disregarded presumed off-target binding (UniProt P26441; HPA tissue IHC).

Boster reagents

Best CNTF / Ciliary neurotrophic factor IHC Antibodies

PB9012 has real IHC images from human paraffin-embedded intestinal and lung cancer sections (catalog IHC image captions); no IF images are supplied (catalog IF image alts).

Real IHC data

Anti-CNTF Antibody ®

Cat # PB9012

View product →

PB9012 is listed for human IHC and WB (catalog applications and reactivity). Its IHC captions show staining in paraffin-embedded human intestinal and lung cancer sections (PB9012 IHC image captions).

Which to pick: For human tissue IHC, choose PB9012 at the listed 0.5–1 μg/mL range; its captions support paraffin sections, and the fixative is unreported (datasheet: IHC dilution; PB9012 IHC image captions). IF/ICC validation is unreported because PB9012 has no listed IF application or IF image (catalog applications; IF image alts). Cross-species reactivity and clonality are unreported; the catalog lists human reactivity and a rabbit host (catalog reactivity, clone field and host).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

UniProt Consortium. UniProt entry P26441 (CNTF_HUMAN, Ciliary neurotrophic factor).
Human Protein Atlas. CNTF tissue IHC expression (reliability: Approved).
Human Protein Atlas. CNTF subcellular location (ICC-IF): Localized to vesicles..
Human Protein Atlas. CNTF antibody validation summary (1 antibodies).
Opposite effects of a high-fat diet and calorie restriction on ciliary neurotrophic factor signaling in the mouse hypothalamus. Frontiers in neuroscience 2013 — PMC3873503.
Action of Administered Ciliary Neurotrophic Factor on the Mouse Dorsal Vagal Complex. Frontiers in neuroscience 2016 — PMC4921504.
Lens Injury Has a Protective Effect on Photoreceptors in the RCS Rat. ISRN ophthalmology 2013 — PMC3914173.
Ciliary neurotrophic factor (CNTF) and its receptor (CNTFRα) signal through MAPK/ERK pathway in human prostate tissues: a morphological and biomolecular study. European journal of histochemistry : EJH 2020 — PMC7586252.
PubMed PMID:1861138 — UniProt-cited evidence.
PubMed PMID:1915374 — UniProt-cited evidence.
PubMed PMID:1840538 — UniProt-cited evidence.