NUMB / Protein numb homolog · IHC design guide

Design Immunohistochemistry for NUMB

Plan paraffin section IHC for NUMB using its observed membranous and cytoplasmic tissue pattern (HPA tissue IHC). Compare high staining in glandular cells with undetected staining in adipocytes, and assess each antibody against appropriate controls (HPA tissue IHC; standard IHC practice).

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

Immunohistochemistry protocol sheet for NUMB (IHC for NUMB): expected localisation Membranous and cytoplasmic staining in most tissues (HPA tissue IHC), antibody PB9301, validated IHC image, and IHC protocol steps — Printable **NUMB** IHC protocol sheet — expected localisation Membranous and cytoplasmic staining in most tissues (HPA tissue IHC), antibody PB9301, controls and protocol steps. Open the full NUMB IHC guide →

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NUMB 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	Membranous and cytoplasmic staining in most tissues (HPA tissue IHC)
	Staining pattern	Glandular cells show membranous and cytoplasmic staining (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet PB9301)
	Positive control ⓘ	Appendix+4 more · see all
	Negative control ⓘ	Adipose tissue+1 more · see all

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific)
	Caveat	Staining has medium consistency with RNA data (HPA tissue IHC)
	Regulation	Expression regulation unreported (UniProt)
	Isoform / epitope	9 isoforms; check epitope coverage for the chosen antibody (UniProt)

Section 1

Recommended NUMB IHC & IF Protocols

The catalog antibody protocol and three published NUMB IHC protocols provide starting conditions for paraffin sections (datasheet: PB9301; PMC5354663; PMC12035580; PMC5308634).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet PB9301)
Fixation	Image fixative and duration unreported (datasheet PB9301); 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 PB9301); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet PB9301)
Primary antibody	Rabbit anti-NUMB, 2-5μg/ml (datasheet PB9301)
Primary incubation	Overnight at 4 °C (datasheet PB9301)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet PB9301)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	NUMB-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Membranous and cytoplasmic expression in most tissues. No signal in the no-primary control.

💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet: PB9301); use each paper’s stated retrieval when reproducing its protocol (PMC5354663; PMC5308634).

Section 2

What Is the Expected NUMB Staining Pattern?

NUMB is expected at cell membranes and in cytoplasm across many tissues, with perinuclear endosomes also described (HPA: tissue IHC; UniProt P49757: localization). Strong staining is reported in appendix and colon glandular cells, bronchial respiratory epithelium, and bone marrow hematopoietic cells (HPA: High). NUMB has no transmembrane segment (UniProt P49757: topology). HPA rates tissue IHC Approved, with medium staining–RNA consistency and external verification pending (HPA: reliability).

What am I looking at on my slide?
Membranous and cytoplasmic staining in appendix or colon glands, bronchial epithelium, or marrow hematopoietic cells.	This fits the reported High staining in those cells and the broader membranous–cytoplasmic tissue profile (HPA: tissue IHC). Judge localization within the identified cells, since neighboring cells need not share their intensity (general IHC practice).
Predominantly nuclear staining, with little membrane or cytoplasmic signal, in an otherwise positive tissue.	That compartment does not match the reported tissue profile or UniProt localization (HPA: tissue IHC; UniProt P49757: localization). Treat an isolated nuclear pattern as suspect; check antibody specificity and detection controls before assigning it to NUMB (general IHC practice).
Strong staining in adipocytes or lymph-node germinal center cells, especially when expected positive cells are weak.	HPA reports NUMB as Not detected in those cell populations (HPA: tissue IHC). Investigate cross-reactivity, endogenous detection activity, and cell identification; an unexpected positive alone cannot establish which explanation applies (general IHC practice).
Diffuse color across cells and surrounding tissue, without a discernible cellular pattern.	This cannot be scored confidently as the reported membranous–cytoplasmic pattern (HPA: tissue IHC). Review the negative detection control, blocking, washes, and chromogen development for nonspecific background (general chromogenic IHC practice).
No staining in appendix or colon glandular cells despite preserved tissue morphology.	Those cells are reported High, so their absence raises a technical or reagent concern (HPA: tissue IHC; general IHC practice). HPA's medium consistency and pending external verification mean a single negative section does not by itself resolve NUMB expression (HPA: reliability).

💡Expected NUMB appearanceCall a section positive when identified glandular, respiratory epithelial, or hematopoietic cells show convincing High membranous–cytoplasmic staining in the listed tissues (HPA: tissue IHC); isolated nuclear color or similar color across tissue and blank spaces is suspect (HPA: tissue IHC; general IHC practice).

How each factor affects the staining
Tissue and cell selection	Use the named High cell populations as expected positives; adipocytes and lymph-node germinal center cells are reported Not detected (HPA: tissue IHC). The label applies to the specified cells, not every cell in that tissue (general IHC practice).
Strength of tissue evidence	HPA calls tissue IHC Approved but notes medium staining–RNA consistency and pending external verification (HPA: reliability). Interpret a disputed pattern with that limitation in view; the supplied record does not establish NUMB-specific fixation sensitivity (HPA: reliability; supplied UniProt P49757 record).
Antibody and epitope interpretation	The supplied list marks CAB011478 IHC Approved and gives HPA002874 no IHC status; it does not provide either epitope (HPA: antibodies). NUMB has 9 listed isoforms and a PID domain at residues 33–193, so isoform coverage cannot be inferred for either antibody (UniProt P49757: isoforms, domain; HPA: antibodies).
IF/ICC Q&A: What localization is reported?	HPA reports mainly cell junctions, with plasma membrane and cytosol also observed; junctional and cytosolic assignments are uncertain, while plasma membrane is supported (HPA: subcellular ICC-IF). Both listed antibodies have Uncertain ICC status, so interpret IF separately from the tissue IHC result (HPA: antibodies).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
No signal in an HPA High cell population.	A failed IHC detection step or an unsuitable antibody setup is possible; the supplied sources give no NUMB-specific fixation explanation (HPA: tissue IHC; general IHC practice).	Check tissue integrity and a known-working positive control, then review the catalog antibody's IHC-P instructions, detection reagents, and run conditions (general IHC practice).
Positive cells stain, but the whole section has a colored haze.	Background can obscure the reported membrane and cytoplasmic pattern (HPA: tissue IHC; general chromogenic IHC practice).	Compare a negative detection control; review blocking, washes, primary-antibody concentration, and chromogen development according to the assay instructions (general chromogenic IHC practice).
The dominant signal is nuclear.	Nuclear staining does not fit the supplied NUMB localization (UniProt P49757: localization; HPA: tissue IHC).	Check whether membrane or cytoplasmic signal appears in HPA High cells, then assess antibody specificity and detection controls before scoring the nuclei (HPA: tissue IHC; general IHC practice).
Adipocytes or germinal center cells stain strongly.	Those populations are reported Not detected; cross-reactivity or endogenous detection activity is possible (HPA: tissue IHC; general IHC practice).	Confirm cell identity and compare negative detection controls with an HPA High population in the same run (HPA: tissue IHC; general IHC practice).
A reported High tissue shows patchy staining.	Cell mix and section variation can complicate scoring; the HPA level refers to named cell populations (HPA: tissue IHC; general IHC practice).	Score the specified cells separately from neighboring cells, compare section morphology and controls, and repeat the assay if the pattern remains unclear (HPA: tissue IHC; general IHC practice).
IF/ICC appears mostly junctional, while tissue IHC looks cytoplasmic.	The modalities have different reported summaries; HPA marks the junctional ICC-IF assignment Uncertain (HPA: subcellular ICC-IF; HPA: tissue IHC).	Interpret each against its own HPA record and validation status; do not use uncertain ICC localization alone to overturn an IHC result (HPA: antibodies, subcellular ICC-IF, tissue IHC).

Source: UniProt P49757 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing NUMB staining

IHC analysis of NUMB using anti-NUMB antibody (PB9301). NUMB was detected in a paraffin-embedded section of human liver 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-NUMB Antibody (PB9301) 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.

PB9301 · IHCIHC analysis of NUMB using anti-NUMB antibody (PB9301). NUMB was detected in a paraffin-embedded section of human liver cancer tissue. Heat

IHC analysis of NUMB using anti-NUMB antibody (PB9301). NUMB was detected in a paraffin-embedded section of human lung 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-NUMB Antibody (PB9301) 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.

PB9301 · IHCIHC analysis of NUMB using anti-NUMB antibody (PB9301). NUMB was detected in a paraffin-embedded section of human lung cancer tissue. Heat m

A01206 · IHCImmunohistochemistry of NUMB in human lung tissue with NUMB antibody at 5 μg/mL.

A01206 · IFImmunofluorescence of NUMB in human lung tissue with NUMB antibody at 20 μg/mL.

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000133961-NUMB/tissue/cerebral+cortex#imid_3643456

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000133961-NUMB/tissue/colon#imid_3643366

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000133961-NUMB/tissue/liver#imid_3643383

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000133961-NUMB/tissue/kidney#imid_3643368

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB011478; green = NUMB, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for NUMB IHC & IF

🧪Run appendix first and expect staining in glandular cells (HPA: appendix glandular cells High). Use adipose tissue adipocytes as the negative tissue (HPA: adipocytes Not detected); on the appendix slide, treat cells without specific staining as internal negatives only after confirming their identity, since the supplied HPA row identifies no negative appendix cell type.

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

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show NUMB in A-431, U-251MG, U2OS, RT-4, RPTEC/TERT1, with annotated localisation: Cell Junctions (uncertain) (HPA subcellular).

Technical controls: Include a no-primary (secondary-only) control, a concentration-matched nonimmune rabbit IgG isotype control, and a validated NUMB-knockout biological negative (caption: rabbit anti-NUMB PB9301; standard IHC controls). Quench endogenous peroxidase and check background in appendix inflammatory cells before interpreting the HRP/DAB signal (caption: HRP/DAB; standard chromogenic IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the exact PB9301 tissue-IHC caption does not state a fixative (caption: fixative not stated). The caption uses heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required (caption: EDTA retrieval). The supplied evidence does not establish whether frozen sections or IF are easier; endogenous peroxidase in appendix inflammatory cells can complicate chromogenic interpretation (standard IHC practice).

HPA tissue IHC evidence for NUMB

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
Appendix	Glandular cells	High	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	High	Protein (IHC)	HPA →
Breast	Glandular cells	High	Protein (IHC)	HPA →
Bronchus	Respiratory epithelial cells	High	Protein (IHC)	HPA →
Cerebellum	Cells in granular layer	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Adipose tissue	Adipocytes	Not detected	Protein (IHC)	HPA →
Lymph node	Germinal center cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced NUMB IHC Tips

Troubleshooting NUMB staining in paraffin sections, with one Q&A on adapting the readout to IF/ICC.

How should I retrieve NUMB when staining paraffin sections?

Start with heat-mediated retrieval in EDTA, pH 8.0 (datasheet PB9301). This is the retrieval used for the catalog antibody in a paraffin section of human liver cancer tissue, followed by 2 μg/ml primary antibody overnight at 4°C (datasheet PB9301). If staining is weak, check heating consistency, section adhesion, and whether the retrieval solution maintained its intended pH before changing conditions (standard IHC practice). Compare any adjusted retrieval condition on adjacent sections with the same detection and development settings, since altered retrieval can change both signal and tissue background (standard IHC practice).

Could fixation explain weak or uneven NUMB staining?

The selected paraffin-section caption does not state a fixative, so NUMB-specific fixation sensitivity for this antibody is unknown (datasheet PB9301). Record fixation method and duration for each specimen, then compare similarly processed sections before attributing a staining difference to NUMB expression (standard IHC practice). If signal varies within a section, inspect tissue preservation and compare central and peripheral regions while keeping retrieval at EDTA, pH 8.0 and primary antibody at 2 μg/ml during the initial comparison (datasheet PB9301; standard IHC practice). A processing-related explanation needs matched specimens or a controlled fixation comparison; the supplied evidence does not establish one for NUMB (datasheet PB9301).

Which NUMB staining compartments should I expect in tissue?

Assess membranous and cytoplasmic staining first: those are the reported patterns across most tissues in the tissue atlas (HPA: tissue IHC profile). Cell-junction staining is reported with uncertain support, plasma-membrane staining with supported evidence, and cytosolic staining with uncertain support in cell imaging (HPA: subcellular profile). NUMB is also annotated at cell and endosome membranes, including AAK1-dependent perinuclear endosomes, despite having no transmembrane segment (UniProt P49757: subcellular location and topology). In chromogenic sections, compare membrane-associated and perinuclear signal against tissue morphology and controls before assigning a fine subcellular location that the section cannot resolve reliably (standard IHC practice).

Can this stain distinguish NUMB isoforms or modified epitopes?

Do not assign a positive chromogenic stain to one NUMB isoform without an antibody epitope and isoform-reactivity map: the record lists 9 isoforms (UniProt P49757: isoforms). The protein contains a PID domain at residues 33–193 and multiple modified residues, including AAK1-associated phosphorylation at threonine 102 (UniProt P49757: domains and modified residues). Those annotations identify possible sources of epitope-dependent behavior but do not establish what this catalog antibody detects (UniProt P49757; datasheet PB9301). For an isoform or phosphorylation claim, obtain epitope-specific validation and compare the result with appropriate independent evidence before interpreting staining intensity as a molecular-state change (standard IHC practice).

How can I adapt NUMB localisation assessment to multiplex IF?

Use the separate IF/ICC guide when planning fluorescence staining; the supplied PB9301 example demonstrates chromogenic staining in a paraffin section, not an IF protocol (datasheet PB9301). Multiplex NUMB with a marker identifying the cell population being assessed, and include single-stain controls to check channel separation (standard IF practice). Choose a fluorophore and detection channel after inspecting tissue autofluorescence, particularly where broad background could obscure membrane or perinuclear signal (standard IF practice; UniProt P49757: subcellular location). Because NUMB has no transmembrane segment and is associated with membranes and endosomes, validate permeabilisation for access to the relevant intracellular-facing epitope once that epitope is known (UniProt P49757: topology and subcellular location; standard IF practice).

What should I check when NUMB DAB staining looks diffuse?

Compare the section with a no-primary control to identify signal from the detection system or endogenous peroxidase, and include a peroxidase-blocking step in the chromogenic workflow (standard IHC practice). The selected example used 10% goat-serum blocking, a peroxidase-conjugated secondary, and DAB development; these details document that example rather than a universal optimum (datasheet PB9301). If background persists, check wash steps and DAB development alongside the primary antibody concentration of 2 μg/ml used in that example (datasheet PB9301; standard IHC practice). Evaluate suspected background against the expected membranous and cytoplasmic pattern and against tissue morphology, rather than treating all brown signal as NUMB (HPA: tissue IHC profile; standard IHC practice).

How should I score NUMB across heterogeneous sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and compartment before scoring, because NUMB tissue staining is reported as membranous and cytoplasmic in most tissues (HPA: tissue IHC profile). For a selected cell population, record the percentage of positive cells and an intensity-based H-score using fixed scoring criteria across sections (standard IHC practice). If spatial distribution matters, report positive-cell density per mm² of viable tissue and document the sampled area (standard IHC practice). Normalise counts to eligible cells or viable tissue area, compare matched compartments, and exclude folds and necrotic regions so processing and tissue composition do not masquerade as expression differences (standard IHC practice).

How can I distinguish credible NUMB staining from artefact?

A credible pattern should agree with the reported membranous and cytoplasmic tissue staining and with the annotated membrane and endosome associations (HPA: tissue IHC profile; UniProt P49757: subcellular location). High staining is reported in appendix glandular cells and bone-marrow hematopoietic cells, whereas adipocytes and lymph-node germinal-center cells are reported as not detected; use those contrasts as context, not absolute controls for this antibody (HPA: tissue IHC profile). Check an unexpected nuclear-only pattern, tissue-edge accentuation, and necrotic areas against intact neighboring tissue and a no-primary control (standard IHC practice). Residual staining in the control can indicate endogenous enzyme activity or detection background, while HPA’s tissue assessment has medium RNA–staining consistency and awaits external verification (standard IHC practice; HPA: reliability assessment).

Boster reagents

Best NUMB / Protein numb homolog IHC Antibodies

Anti-NUMB antibodies have human tissue IHC images (catalog IHC image captions), and A01206 also has a human lung-tissue IF image (catalog IF image caption). Both list Human, Mouse, and Rat reactivity (catalog reactivity).

Real IHC data

Anti-NUMB Antibody ®

Cat # PB9301

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Real IHC data Immunohistochemistry of NUMB in human lung tissue with NUMB antibody at 5 μg/mL.

Anti-Protein numb homolog NUMB Antibody

Cat # A01206

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PB9301 shows IHC staining in paraffin-embedded human liver cancer tissue (PB9301 IHC image caption). A01206 shows IHC staining and IF in human lung tissue (A01206 IHC and IF image captions).

Which to pick: For paraffin-section IHC, choose PB9301: its caption documents antigen retrieval and chromogenic detection in a paraffin-embedded section; the fixative is unreported (PB9301 IHC image caption). For IF, choose A01206, which has a human lung-tissue IF image; ICC is not listed, and its IHC caption does not report a fixative (A01206 IF image caption; catalog applications; A01206 IHC image caption). For cross-species planning, both list Human, Mouse, and Rat reactivity, while PB9301 additionally lists Monkey; the supplied IHC images document human samples only (catalog reactivity; catalog IHC image captions).

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

References

UniProt Consortium. UniProt entry P49757 (NUMB_HUMAN, Protein numb homolog).
Human Protein Atlas. NUMB tissue IHC expression (reliability: Approved).
Human Protein Atlas. NUMB subcellular location (ICC-IF): Mainly localized to the cell junctions. In addition localized to the plasma membrane and cytosol..
Human Protein Atlas. NUMB antibody validation summary (2 antibodies).
Expression of Notch1 and Numb in small cell lung cancer. Oncotarget 2017 — PMC5354663.
Expression of NUMB Protein and Its Prognostic Significance in Endometrial Cancer: A Retrospective Cohort Study. Cureus 2025 — PMC12035580.
NUMB negatively regulates the epithelial-mesenchymal transition of triple-negative breast cancer by antagonizing Notch signaling. Oncotarget 2016 — PMC5308634.
Loss of Numb promotes hepatic progenitor expansion and intrahepatic cholangiocarcinoma by enhancing Notch signaling. Cell death & disease 2021 — PMC8526591.
PubMed PMID:9303539 — UniProt-cited evidence.
PubMed PMID:10468633 — UniProt-cited evidence.
PubMed PMID:21486681 — UniProt-cited evidence.