CLTB / Clathrin light chain B · IHC design guide

Design Immunohistochemistry for CLTB

Plan chromogenic CLTB IHC in paraffin sections using the catalog antibody at 2–5 μg/ml (datasheet A09071-1). Expect often granular cytoplasmic staining (HPA tissue IHC); epididymis and small intestine glandular cells show high staining and can guide positive-control selection (HPA tissue IHC).

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

Immunohistochemistry protocol sheet for CLTB (IHC for CLTB): expected localisation Cytoplasmic, often granular (HPA tissue IHC), antibody A09071-1, validated IHC image, and IHC protocol steps — Printable **CLTB** IHC protocol sheet — expected localisation Cytoplasmic, often granular (HPA tissue IHC), antibody A09071-1, controls and protocol steps. Open the full CLTB IHC guide →

Navigate to: Protocols Staining pattern Controls Antibodies

CLTB 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, often granular (HPA tissue IHC)
	Staining pattern	Cytoplasmic staining, often granular (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A09071-1)
	Positive control ⓘ	Epididymis+4 more · see all
	Negative control ⓘ	Caudate

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Keep fixation consistent across sections (standard IHC practice; not target-specific). Selected-image fixative and duration unreported (datasheet A09071-1); verify before use.
	Caveat	Staining and RNA show only medium agreement (HPA tissue IHC)
	Regulation	No expression regulator annotated (UniProt)
	Isoform / epitope	2 isoforms, brain and non-brain; epitope map unspecified (UniProt)

Section 1

Recommended CLTB IHC & IF Protocols

The catalog antibody’s IHC-P protocol (datasheet: A09071-1) is accompanied by one published paraffin-section protocol (PMC10157171).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human endometrial cancer tissue; fixative not specified (datasheet A09071-1)
Fixation	Image fixative and duration unreported (datasheet A09071-1); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat retrieval: EDTA pH 8.0 (datasheet A09071-1); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A09071-1)
Primary antibody	Rabbit anti-CLTB, 2-5 μg/ml (datasheet A09071-1)
Primary incubation	Overnight at 4 °C (datasheet A09071-1)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet A09071-1)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	CLTB-positive staining in glandular cells of epididymis (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression often with a granular pattern. No signal in the no-primary control.

💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval (datasheet: A09071-1); the published excerpt does not specify retrieval (PMC10157171).

Section 2

What Is the Expected CLTB Staining Pattern?

CLTB should appear mainly as granular cytoplasmic staining in paraffin sections, consistent with its location on the cytoplasmic face of coated pits and vesicles and its lack of a transmembrane segment (UniProt P09497 topology; HPA tissue IHC profile). Epididymal and small-intestinal glandular cells are strong reference positives (HPA: High). HPA rates its tissue IHC profile Approved, with medium agreement between staining and RNA data; external verification is pending (HPA tissue IHC reliability).

What am I looking at on my slide?
Granular cytoplasmic staining in epididymal or small-intestinal glandular cells, with a clear counterstain.	This fits the strongest listed tissue patterns (HPA: High in both cell populations) and the expected vesicle-associated compartment (UniProt P09497 subcellular location). Judge signal against nearby tissue and a matched control; a positive result alone does not establish antibody specificity (general IHC practice).
Predominantly nuclear staining or a sharply defined membrane-only pattern.	This conflicts with the general granular cytoplasmic tissue profile (HPA tissue IHC profile). CLTB also associates with coated pits at the plasma membrane, so limited peripheral signal is plausible (UniProt P09497 subcellular location). Treat a dominant unexpected compartment as a reason to check controls and antibody specificity, not as a new CLTB location.
Strong staining in cells listed as weak or undetected, while expected positive cells do not stain.	Compare the exact cell population: parathyroid glandular cells, myocytes and adipocytes are listed Low, and caudate glial cells Not detected (HPA tissue IHC). This mismatch raises possible cross-reactivity or endogenous detection activity; tissue identity, morphology and controls must resolve it (general IHC practice).
Uniform colour across cells and extracellular areas, obscuring cytoplasmic granules.	A diffuse field does not match the reported granular cytoplasmic profile (HPA tissue IHC profile). Review the no-primary control, blocking, washing and detection system for background (general IHC practice). Do not score a uniformly coloured field as CLTB positive solely because the tissue is listed as expressing CLTB.
No convincing signal in epididymal or small-intestinal glandular cells.	These are useful positive references because both are listed High (HPA tissue IHC). First check that the section contains the named cells and that the detection controls worked (general IHC practice). An absent stain after valid controls may reflect assay performance; it cannot by itself overturn the HPA tissue pattern.

💡Expected CLTB appearanceCall positive when glandular cells show clear, predominantly granular cytoplasmic staining, strongest in the listed High tissues (HPA tissue IHC); dominant nuclear colour or diffuse colour outside cells is suspect against that profile (HPA tissue IHC profile; general IHC practice).

How each factor affects the staining
Compartment and topology	CLTB is on the cytoplasmic face of coated pits and vesicles, with no transmembrane segment (UniProt P09497 topology). Expect cytoplasmic granules in tissue (HPA tissue IHC profile); topology does not specify a retrieval condition.
Tissue and cell selection	Epididymal and small-intestinal glandular cells are High; several other listed populations are Medium or Low (HPA tissue IHC). Select a named positive population and score that population, rather than treating an entire section as uniformly positive.
Isoforms and epitope coverage	UniProt lists Brain and Non-brain isoforms (UniProt P09497 isoforms). Their existence alone does not establish which isoform an antibody detects. Check the chosen antibody's stated epitope coverage before interpreting a regional difference as isoform-specific.
IHC evidence strength	The tissue profile is Approved with medium staining–RNA agreement and pending external verification (HPA tissue IHC reliability). The listed mouse monoclonal has IHC Approved status (HPA antibodies: CAB003692). These labels support cautious use of the pattern, not independent confirmation of every stained cell.
What should IF/ICC show?	Predominantly vesicular signal, with additional plasma-membrane and cytosolic localisation (HPA subcellular ICC-IF). This is a compartment cross-check, not a paraffin IHC result; the listed rabbit polyclonal is ICC Supported, with no IHC status supplied (HPA antibodies: HPA036458).
Retrieval and fixation evidence	No CLTB-specific retrieval setting or fixation sensitivity is reported in the supplied UniProt and HPA records. If signal is weak, optimise retrieval against a named positive tissue and controls as a general IHC practice; do not infer a CLTB-specific fixation effect.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Expected positive glandular cells remain blank.	The assay may have failed, or the named cell population may be absent from the section (general IHC practice).	Confirm cell identity and run a valid detection control; compare with epididymal or small-intestinal glandular cells listed High (HPA tissue IHC). Then optimise the general IHC workflow without assuming CLTB-specific fixation sensitivity.
Colour is widespread, including areas without cells.	Background from blocking, washing or the detection system may obscure the reported granular cytoplasmic pattern (general IHC practice; HPA tissue IHC profile).	Inspect a no-primary control and adjust blocking, washing or detection conditions as indicated (general IHC practice). Reassess only cell-associated signal after background is controlled.
The main signal is nuclear.	A nuclear-dominant pattern is inconsistent with the reported tissue profile and vesicle/coated-pit location (HPA tissue IHC profile; UniProt P09497 subcellular location).	Check morphology, counterstain and negative controls; repeat with a validated antibody if needed (general IHC practice). Do not score isolated nuclear colour as the expected CLTB pattern.
Unexpectedly strong signal appears in a listed weak or negative cell population.	Possible cross-reactivity or endogenous detection activity; the mismatch alone does not distinguish them (HPA tissue IHC levels; general IHC practice).	Verify the cell type, compare a no-primary control and inspect a named High positive on the same run (HPA tissue IHC; general IHC practice). Report any unresolved discordance.
Peripheral staining appears alongside cytoplasmic granules.	Coated pits occur at the plasma membrane, and ICC-IF reports an additional plasma-membrane location (UniProt P09497 subcellular location; HPA subcellular ICC-IF).	Assess whether granular cytoplasmic signal still dominates the IHC pattern (HPA tissue IHC profile). Investigate a membrane-only result with controls before assigning it to CLTB (general IHC practice).
Two antibodies yield different tissue patterns.	HPA lists separate application assessments: CAB003692 is IHC Approved, while HPA036458 is ICC Supported with no IHC status supplied (HPA antibodies).	For paraffin IHC, interpret each antibody against its own application evidence and a named High tissue (HPA antibodies; HPA tissue IHC). Do not transfer an ICC validation label to IHC.

Source: UniProt P09497 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing CLTB staining

IHC analysis of CLTB using anti-CLTB antibody (A09071-1). CLTB was detected in a paraffin-embedded section of human endometrial cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-CLTB Antibody (A09071-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

A09071-1 · IHCIHC analysis of CLTB using anti-CLTB antibody (A09071-1). CLTB was detected in a paraffin-embedded section of human endometrial cancer tissu

IHC analysis of CLTB using anti-CLTB antibody (A09071-1). CLTB was detected in a paraffin-embedded section of human pancreas cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-CLTB Antibody (A09071-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

A09071-1 · IHCIHC analysis of CLTB using anti-CLTB antibody (A09071-1). CLTB was detected in a paraffin-embedded section of human pancreas cancer tissue.

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000175416-CLTB/tissue/cerebral+cortex#imid_1267644

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000175416-CLTB/tissue/colon#imid_1267586

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000175416-CLTB/tissue/liver#imid_1267579

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000175416-CLTB/tissue/kidney#imid_1267580

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA036458; green = CLTB, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for CLTB IHC & IF

🧪Run epididymis first and expect glandular cells to stain (HPA: High in epididymis glandular cells). Use caudate glial cells as the negative tissue comparator (HPA: Not detected in caudate glial cells); on the epididymis slide, assess nonglandular cells for background without assuming they are CLTB-negative (HPA: glandular cells High).

Positive control tissue: Epididymis (Glandular cells, HPA High)

Negative control tissue: Caudate (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CLTB in A-431, MCF-7, U2OS, NIH 3T3, with annotated localisation: Vesicles (supported) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control and a concentration-matched rabbit IgG isotype control (selected-SKU caption: rabbit primary antibody). Use CLTB knockout material, if available, as a biological specificity control; quench endogenous peroxidase and inspect background in the epididymis section before interpreting DAB staining (selected-SKU caption: peroxidase detection with DAB).

⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU caption: fixative not stated). Heat retrieval in EDTA at pH 8.0 is documented for the catalog antibody, but a requirement for that retrieval method is unreported (selected-SKU caption: heat-mediated EDTA retrieval). Frozen-section performance and whether it is easier than paraffin IHC are unreported; ICC-IF images support vesicular localization, while any epididymis-specific staining artefact is unreported (HPA subcellular: vesicles supported; HPA: epididymis glandular cells High).

HPA tissue IHC evidence for CLTB

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. 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
Epididymis	Glandular cells	High	Protein (IHC)	HPA →
Small intestine	Glandular cells	High	Protein (IHC)	HPA →
Adrenal gland	Glandular cells	Medium	Protein (IHC)	HPA →
Appendix	Glandular cells	Medium	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	Medium	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Caudate	Glial cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced CLTB IHC Tips

Troubleshoot CLTB staining in paraffin sections by checking retrieval, compartment, controls, and scoring before interpreting differences between samples.

Which retrieval conditions should I try first for CLTB in paraffin sections?

Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin CLTB IHC (datasheet A09071-1). The catalog antibody stained a human endometrial cancer section after that retrieval, followed by 2 μg/ml primary antibody overnight at 4°C (datasheet A09071-1). If staining is weak, vary heating duration on adjacent sections while keeping the antibody concentration, detection chemistry, and exposure to DAB consistent (standard IHC practice). Compare granular cytoplasmic staining with background and tissue preservation; excess heat can damage morphology and make apparent signal difficult to assign to cells (standard IHC practice).

Could fixation explain weak or uneven CLTB staining?

CLTB-specific fixation sensitivity is unknown because the selected paraffin-section caption does not state a fixative (datasheet A09071-1). Record each specimen's fixative and fixation duration, and compare sections processed alike before attributing intensity differences to CLTB abundance (standard IHC practice). Include a section stained with the documented pH 8.0 EDTA retrieval and 2 μg/ml primary antibody as a procedural reference (datasheet A09071-1). Uneven fixation or processing can alter morphology and staining across a section, so inspect the tissue before scoring (standard IHC practice). Do not infer a CLTB-specific fixation effect from its cytoplasmic-facing location or phosphorylation sites (UniProt P09497).

What staining pattern is credible for CLTB in chromogenic IHC?

Expect predominantly cytoplasmic, often granular staining in tissue sections (HPA: tissue IHC profile), consistent with CLTB on the cytoplasmic face of coated pits and vesicles (UniProt P09497 subcellular location). Vesicles are the supported main location, with additional plasma-membrane and cytosolic signal reported in cell imaging (HPA: subcellular). Assess whether DAB granules lie within intact cell boundaries and recur in comparable cells, using a light counterstain to identify nuclei (standard IHC practice). Strong isolated nuclear staining or diffuse deposit outside cells warrants a detection and background check before it is scored as CLTB (UniProt P09497 subcellular location; standard IHC practice).

Can CLTB isoforms or epitope accessibility change my IHC result?

CLTB has Brain and Non-brain isoforms, but the supplied antibody caption does not identify its epitope or establish isoform selectivity (UniProt P09497 isoforms; datasheet A09071-1). Its 229-residue chain has no annotated transmembrane segment, and CLTB faces the cytoplasm on coated pits and vesicles (UniProt P09497 topology and subcellular location). Consequently, a staining difference alone cannot identify an isoform or a modified residue; documented phosphorylation and acetylation sites establish neither antibody sensitivity nor tissue-specific staining (UniProt P09497 modified residues). If isoform discrimination matters, obtain epitope mapping or an independently validated isoform-specific reagent and compare matched sections under identical retrieval conditions (standard IHC practice).

How should I plan a complementary CLTB IF experiment?

For a complementary IF experiment, pair CLTB with a marker for the cell type under study and choose fluorophores whose emission is distinguishable from tissue autofluorescence (standard IF practice). CLTB lies on the cytoplasmic face of coated pits and vesicles, so plan permeabilisation to let antibody reach an intracellular epitope (UniProt P09497 subcellular location; standard IF practice). Compare CLTB puncta with the cell marker and include single-stain controls to assess bleed-through and autofluorescence (standard IF practice). Vesicular localisation is supported in cell imaging, but that observation alone does not establish how the IHC-validated catalog antibody will perform in IF (HPA: subcellular; datasheet A09071-1).

How can I separate CLTB granules from chromogenic background?

First compare the complete stain with a section omitting primary antibody, while retaining the same secondary reagent and DAB development (standard IHC practice). The documented CLTB workflow used 10% goat-serum block, 2 μg/ml primary antibody overnight at 4°C, and peroxidase-conjugated secondary antibody for 30 minutes at 37°C (datasheet A09071-1). Add a peroxidase block and monitor DAB development as general chromogenic IHC controls (standard IHC practice). CLTB may look granular in cytoplasm, so judge a suspected deposit against intact cell boundaries, the no-primary control, and similarly processed sections before changing primary concentration (HPA: tissue IHC profile; standard IHC practice).

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

Define the tissue compartment and cell population before scoring, then use one consistent measure such as an H-score, percentage of positive cells, or positive-cell density per mm² (standard IHC practice). If using an H-score, record the percentage of cells at each intensity category and apply the same thresholds to every slide (standard IHC practice). Normalise counts or density to viable, evaluable tissue area or the number of eligible cells, and exclude folds, edges, and necrotic regions (standard IHC practice). Report retrieval, antibody concentration, DAB development, and image-analysis thresholds alongside results; the documented antibody example used pH 8.0 EDTA and 2 μg/ml primary antibody (datasheet A09071-1).

When does an apparent CLTB-positive section need further validation?

Give most weight to granular cytoplasmic staining within intact cells, the general tissue pattern reported for CLTB (HPA: tissue IHC profile). Staining confined to nuclei, cut edges, necrotic areas, or extracellular debris conflicts with CLTB's coated-pit and vesicle localisation and needs control review (UniProt P09497 subcellular location; standard IHC practice). Check a no-primary section and the peroxidase block when DAB appears broadly distributed, because endogenous enzyme activity can mimic chromogenic signal (standard IHC practice). Use the sampled cell population in context: HPA reports high staining in epididymal and small-intestinal glandular cells, while its tissue IHC reliability is Approved with medium RNA agreement and pending external verification (HPA: tissue IHC).

Boster reagents

Best CLTB / Clathrin light chain B IHC Antibodies

A09071-1 has real IHC images from human endometrial and pancreas cancer sections and mouse and rat cerebellum sections (catalog IHC captions). No IF image is supplied (catalog payload).

Real IHC data

Anti-CLTB Antibody ®

Cat # A09071-1

View product →

A09071-1 is listed for IHC in human, mouse and rat (catalog applications and reactivity). Its own IHC captions document staining in paraffin sections of human endometrial and pancreas cancer, mouse cerebellum and rat cerebellum (catalog IHC captions).

Which to pick: Choose A09071-1 for paraffin-section IHC; its captions report staining at 2 μg/ml after EDTA pH 8.0 retrieval, but do not report the fixative (A09071-1 IHC captions). The same SKU has captioned IHC examples across human, mouse and rat (A09071-1 IHC captions). There is no IF/ICC-validated SKU in this payload, so an IF/ICC choice cannot be supported here (catalog applications; catalog IF images).

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

References

UniProt Consortium. UniProt entry P09497 (CLCB_HUMAN, Clathrin light chain B).
Human Protein Atlas. CLTB tissue IHC expression (reliability: Approved).
Human Protein Atlas. CLTB subcellular location (ICC-IF): Mainly localized to vesicles. In addition localized to the plasma membrane and cytosol..
Human Protein Atlas. CLTB antibody validation summary (2 antibodies).
Clathrin Light Chain B Drives Hepatocellular Carcinoma Progression Through Dual Mechanisms: Small Extracellular Vesicle-Mediated Angiogenesis and the NF-κB-PCLAF Signaling Axis. Advanced science (Weinheim, Baden-Wurttemberg, Germany) 2025 — PMC12622436.
A novel 25-ferroptosis-related gene signature for the prognosis of gliomas. Frontiers in oncology 2023 — PMC10157171.
PubMed PMID:3267234 — UniProt-cited evidence.
PubMed PMID:15489334 — UniProt-cited evidence.
PubMed PMID:11889126 — UniProt-cited evidence.