CNTF / Ciliary neurotrophic factor · IHC design guide

Design Immunohistochemistry for CNTF

Plan CNTF paraffin-section IHC using the high staining reported in caudate neurons and cerebellar Purkinje cells (HPA tissue IHC). Interpret staining alongside the probable cytoplasmic precursor and possible release 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 Probable cytoplasmic precursor; secreted CNTF (UniProt), antibody PB9012, validated IHC image, and IHC protocol steps — Printable **CNTF** IHC protocol sheet — expected localisation Probable cytoplasmic precursor; secreted CNTF (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	Probable cytoplasmic precursor; secreted CNTF (UniProt)
	Staining pattern	High staining in caudate neurons and cerebellar 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	Release from damaged cells may alter tissue staining (UniProt)
	Regulation	Expression regulation not annotated (UniProt)
	Isoform / epitope	No annotated isoforms; one 1–200 chain (UniProt)

Section 1

Recommended CNTF IHC & IF Protocols

The catalog antibody’s paraffin IHC protocol (datasheet PB9012) is accompanied by published chromogenic CNTF protocols for mouse brain and prostate tissue (PMC3873503; PMC4921504; 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 retrieval at pH 6 (datasheet PB9012; PMC7586252: methods).

Section 2

What Is the Expected CNTF Staining Pattern?

CNTF staining should be assessed chiefly in CNS cells: HPA reports high IHC staining in caudate neuronal cells and cerebellar Purkinje cells, and medium staining in cortical neuronal cells (HPA tissue IHC, Approved). A predominantly cytoplasmic signal is consistent with CNTF's annotated localization and lack of a transmembrane segment (UniProt P26441). HPA's Approved tissue assessment notes that presumed off-target binding was observed and disregarded (HPA tissue IHC).

What am I looking at on my slide?
Clear chromogenic signal in caudate neuronal cells or cerebellar Purkinje cells, with lighter signal in cortical neuronal cells.	This matches the reported high staining in caudate and cerebellum and medium staining in cortex (HPA tissue IHC). Judge intensity in the named cells; HPA's entries do not assign one staining level to every cell in those tissues (HPA tissue IHC).
Predominantly cytoplasmic staining in the expected cells, possibly with a punctate appearance.	Cytoplasmic localization is annotated for CNTF (UniProt P26441); vesicles are the approved ICC-IF location (HPA subcellular). Vesicle-scale detail may be difficult to resolve in chromogenic paraffin sections (general IHC practice), so puncta are supportive rather than required.
Strong, isolated nuclear staining dominates while the expected cytoplasmic pattern is absent.	An isolated nuclear pattern does not match the annotated cytoplasmic location or approved vesicular ICC-IF location (UniProt P26441; HPA subcellular). Treat it as suspect and check controls before assigning it to CNTF (general IHC practice).
Strong staining appears in HPA-listed negative cells, such as adipocytes in adipose tissue or hematopoietic cells in bone marrow.	Those specific cell and tissue combinations were reported as not detected (HPA tissue IHC). Consider cross-reactivity or endogenous chromogen activity, and inspect controls (general IHC practice). HPA's negative entries do not rule out staining in every cell of those organs.
Diffuse color covers tissue broadly, or expected CNS cells show no signal.	Broad diffuse color obscures comparison with HPA's cell-specific pattern (HPA tissue IHC; general IHC practice). An absent signal in caudate neuronal cells or Purkinje cells conflicts with their reported high staining, but first assess slide quality and assay controls (HPA tissue IHC; general IHC practice).

💡Expected CNTF appearanceA convincing positive is predominantly cytoplasmic staining in caudate neuronal cells or cerebellar Purkinje cells at the reported high level, with cortical neuronal cells reported at medium; dominant nuclear or widespread negative-cell staining is suspect (UniProt P26441; HPA tissue IHC; HPA subcellular).

How each factor affects the staining
Tissue and cell choice	Caudate neuronal cells and cerebellar Purkinje cells are high-staining reference populations; cortical neuronal cells are medium (HPA tissue IHC). Compare named cell populations within their tissue context.
Subcellular interpretation	CNTF is annotated as cytoplasmic and secreted, with no transmembrane segment (UniProt P26441). HPA approves vesicular localization by ICC-IF; this informs localization but does not establish a required punctate IHC pattern (HPA subcellular).
Protein and RNA evidence	Bone marrow has tissue-enhanced RNA specificity, while bone marrow hematopoietic cells are reported as not detected by tissue IHC (HPA tissue IHC). Do not substitute the RNA category for an expected protein-staining result.
Antibody validation	HPA019654 is listed as Approved for IHC, and the tissue profile is Approved with presumed off-target binding observed and disregarded (HPA antibodies; HPA tissue IHC). Approval supports use of the reported pattern but does not make every new signal specific.
IF/ICC Q: What location is reported?	A: Vesicles are the approved main ICC-IF location, with images listed for A-431, U-251MG and U2OS (HPA subcellular). This is an IF/ICC observation, not an IHC-P protocol or a prediction of staining intensity in those cell lines.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
No chromogenic signal in caudate neuronal cells or cerebellar Purkinje cells.	Both are reported at high staining, so the result needs an assay check before biological interpretation (HPA tissue IHC).	Review tissue preservation, assay controls, antibody incubation and detection steps using the chosen IHC-P procedure (general IHC practice). No CNTF-specific fixation or retrieval sensitivity is established by the supplied sources.
Strong staining is confined to nuclei.	That distribution conflicts with cytoplasmic CNTF annotation and approved vesicular ICC-IF localization (UniProt P26441; HPA subcellular).	Check negative controls and compare with the expected CNS cell pattern before scoring the nuclear signal as specific (HPA tissue IHC; general IHC practice).
Adipocytes in adipose tissue or hematopoietic cells in bone marrow stain strongly.	HPA reports these particular cell populations as not detected; cross-reactivity or endogenous detection activity is possible (HPA tissue IHC; general IHC practice).	Review an appropriate negative control and detection-system controls, then compare cell-level staining with a reported CNS positive (HPA tissue IHC; general IHC practice).
Diffuse chromogen obscures cell boundaries throughout the section.	Nonspecific background or endogenous detection activity can hinder localization in chromogenic IHC (general IHC practice).	Inspect the negative control; review blocking, washes and detection conditions according to the selected IHC-P procedure (general IHC practice). Reassess only where individual cells can be distinguished.
Bone marrow appears negative despite its tissue-enhanced RNA category.	The RNA category and the reported protein IHC result differ: hematopoietic cells were not detected by IHC (HPA tissue IHC).	Use the cell-specific IHC observation when setting staining expectations; evaluate assay performance in reported high-staining CNS cells (HPA tissue IHC).
Cytoplasmic staining is present without resolvable vesicular puncta.	Vesicles are an approved ICC-IF location, whereas the chromogenic paraffin readout may not resolve fine puncta (HPA subcellular; general IHC practice).	Score the named cell populations and overall cytoplasmic distribution against the tissue IHC profile; do not require visible puncta as the sole criterion (HPA tissue IHC; UniProt P26441; 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 and score Purkinje cells, which show High CNTF staining (HPA: Cerebellum, Purkinje cells, High). Use adipose tissue as the negative tissue, scoring adipocytes reported as Not detected (HPA: Adipose tissue, adipocytes, Not detected); compare other cells on the cerebellum slide with Purkinje cells as an internal background reference, without assuming those cells are CNTF-negative.

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 slide and a rabbit isotype control matched to the primary antibody’s immunoglobulin class (selected IHC caption: rabbit anti-CNTF); use CNTF-knockout tissue, if available, as a biological specificity control. Quench endogenous peroxidase for DAB detection and check endogenous biotin background when using the caption’s biotinylated secondary and streptavidin–biotin detection (selected IHC caption: biotinylated secondary, SABC, DAB).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected PB9012 paraffin-section caption does not state a fixative (selected IHC caption: fixative not stated). That caption used citrate retrieval at pH 6 for 20 minutes, but it does not establish whether retrieval is required (selected IHC caption: heat-mediated citrate retrieval). The supplied evidence does not establish that frozen sections or IF are easier; if assessing IF in cerebellum, check neuronal autofluorescence with an unstained section (standard IF practice; HPA: Cerebellum, Purkinje cells, High).

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

Troubleshoot CNTF staining in paraffin sections by checking retrieval, cell identity, localisation and controls before comparing signal intensity across samples.

Which retrieval conditions should I try first for CNTF chromogenic IHC?

Start with heat mediated retrieval in citrate buffer at pH 6 for 20 minutes (datasheet PB9012). That condition preceded CNTF staining in a paraffin section of human intestinal cancer tissue, although the caption does not report the fixative (datasheet PB9012). If signal is weak, compare a modest change in heating time on matched sections while keeping antibody concentration and detection constant (standard IHC practice). Include a neuronal region with reported CNTF staining when available, such as caudate neuronal cells, to judge whether retrieval worked (HPA: High in caudate neuronal cells). Excessive heating can damage morphology, so assess staining and tissue preservation together (standard IHC practice).

Could fixation explain weak CNTF staining in my paraffin sections?

The selected paraffin tissue caption does not state a fixative, so target specific CNTF sensitivity to fixation is unknown (datasheet PB9012). Do not infer fixation tolerance from the reported tissue staining pattern or CNTF protein features (HPA: tissue IHC; UniProt P26441). Record fixative, fixation duration, processing schedule and section age for each specimen before comparing stain intensity (standard IHC practice). If fixation varies, stain matched sections in the same run and compare morphology and a control section alongside CNTF (standard IHC practice). Optimise retrieval on those sections before attributing a weak result to fixation, since retrieval and detection also affect chromogenic intensity (standard IHC practice).

Where should CNTF staining appear within positive cells?

Evaluate intracellular CNTF staining against the annotated cytoplasmic precursor and the reported secreted protein, without requiring a continuous plasma membrane outline (UniProt P26441 subcellular location; UniProt P26441 topology). Vesicular localisation was approved in cell imaging, but that observation alone does not establish a punctate pattern for paraffin tissue IHC (HPA: subcellular localisation). Compare staining within morphologically identified neuronal cells where reference tissue is available; caudate neurons and cerebellar Purkinje cells have reported high staining (HPA: tissue IHC). A strong nuclear only pattern needs independent validation because the supplied localisation evidence does not identify a nuclear pool (UniProt P26441 subcellular location; HPA: subcellular localisation). Score localisation separately from intensity (standard IHC practice).

How do CNTF processing and epitope uncertainty affect stain interpretation?

CNTF has one annotated chain spanning residues 1–200, with no annotated isoforms, signal peptide, propeptide or glycosylation sites (UniProt P26441 processing; UniProt P26441 isoforms; UniProt P26441 glycosylation). The record also lists no transmembrane segment, so a sharp membrane rim should prompt scrutiny rather than automatic CNTF assignment (UniProt P26441 topology). The supplied antibody caption does not identify the epitope, so staining cannot be assigned to a particular end of the protein from these data (datasheet PB9012). Compare a second antibody with a documented distinct epitope, if available, and require concordant cell identity and localisation (standard IHC practice). Do not interpret variable staining as an isoform shift without independent evidence (UniProt P26441 isoforms).

How can IF help check an ambiguous CNTF IHC pattern?

Use IF as an orthogonal localisation check, with a neuronal cell marker to identify the cells expected to stain in reference CNS tissue (HPA: High in caudate neuronal cells and cerebellar Purkinje cells). Choose a fluorophore channel separated from the specimen’s autofluorescence, and examine unstained and single stain controls before interpreting overlap (standard IF practice). For an intracellular epitope, compare mild permeabilisation with an unpermeabilised condition; the CNTF precursor is probably cytoplasmic, while the antibody epitope is unspecified (UniProt P26441 subcellular location; datasheet PB9012). Vesicular IF localisation has been reported, but it does not by itself validate the catalog antibody’s IF performance (HPA: subcellular localisation). Keep IF findings separate from chromogenic IHC scores (standard analysis practice).

What should I check when CNTF DAB staining is widespread?

First inspect a section processed without primary antibody to assess secondary reagent, endogenous enzyme and chromogen background (standard IHC practice). The selected method used 10% goat serum, biotinylated anti rabbit secondary antibody, a streptavidin biotin complex and DAB, so blocking and detection controls matter for that workflow (datasheet PB9012). Include an appropriate peroxidase block and assess endogenous biotin interference when using biotin based detection (standard chromogenic IHC practice). Compare diffuse staining with expected cell patterns; HPA reports mainly CNS expression and notes presumed off target binding in its tissue assessment (HPA: tissue IHC). Review section edges and damaged areas before calling broad brown signal CNTF positive (standard IHC practice).

How should I quantify CNTF staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define a cell population and region before scoring, then report the percentage of positive cells and an intensity weighted H score for that population (standard IHC quantification practice). Alternatively, count positive cells per mm² when tissue architecture makes area based comparison more reliable (standard IHC quantification practice). Normalise cell counts to the analysed viable tissue area, and keep staining threshold, imaging settings and batch controls consistent across samples (standard IHC quantification practice). Score neuronal cells separately from other cells in CNS reference sections, since reported positive populations include caudate neurons and cerebellar Purkinje cells (HPA: tissue IHC). Record localisation and exclude folds, necrosis and edge artefacts from the analysed area (standard IHC practice).

When is a CNTF positive stain credible rather than an artefact?

A credible result follows a reproducible cell specific pattern and agrees with an appropriate control section, while allowing for the cytoplasmic precursor and possible extracellular CNTF (standard IHC practice; UniProt P26441 subcellular location). High staining is reported in caudate neuronal cells and cerebellar Purkinje cells, whereas HPA also flags presumed off target binding in its tissue assessment (HPA: tissue IHC). Scrutinise nuclear only staining, a continuous membrane rim or signal confined to tissue edges and necrotic areas before assigning it to CNTF (UniProt P26441 subcellular location; UniProt P26441 topology; standard IHC practice). Check a no primary control and the peroxidase block when brown signal could reflect endogenous enzyme activity (standard chromogenic IHC practice). Resolve discordant patterns with an independent antibody or orthogonal assay (standard validation practice).

Boster reagents

Best CNTF / Ciliary neurotrophic factor IHC Antibodies

PB9012 has real CNTF IHC images from human paraffin sections of intestinal and lung cancer tissues (PB9012 IHC captions). No IF images are supplied (PB9012 catalog IF image alts).

Real IHC data

Anti-CNTF Antibody ®

Cat # PB9012

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PB9012 is listed for human IHC and WB (PB9012 catalog applications/reactivity). Its IHC captions show CNTF staining in paraffin sections of human intestinal and lung cancer tissues (PB9012 IHC captions).

Which to pick: Choose PB9012 for human tissue IHC: it is a rabbit antibody listed for IHC, with a recommended concentration of 0.5–1 μg/ml and paraffin-section images (PB9012 catalog host/applications/IHC dilution; PB9012 IHC captions). There is no supported IF/ICC or cross-species choice in this payload: PB9012 lists human reactivity and no IF images or IF dilution (PB9012 catalog reactivity/IF image alts/IF dilution). The captions document paraffin sections but do not report the fixative (PB9012 IHC captions).

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.