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- Table of Contents
NUMB / Protein numb homolog · IHC design guide
Design Immunohistochemistry for NUMB
Plan paraffin-section NUMB IHC around its membranous and cytoplasmic tissue staining (HPA tissue IHC). High staining in glandular cells supports positive-reference selection (HPA tissue IHC), while 9 isoforms warrant epitope review (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
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 | Strong glandular-cell staining; membrane and cytoplasm (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 conditions consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | IHC staining has medium concordance with RNA data (HPA tissue IHC) | |
| Regulation | Staining-linked expression regulation is unreported (UniProt) | |
| Isoform / epitope | 9 isoforms; check epitope conservation across forms (UniProt) |
Section 1
Recommended NUMB IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet: PB9301) with 3 published NUMB IHC protocols (PMC5354663; PMC12035580; PMC5308634).
| 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 heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet: PB9301); the published citrate conditions apply to their respective antibodies (PMC5354663; PMC5308634).
Section 2
What Is the Expected NUMB Staining Pattern?
NUMB should show membranous and cytoplasmic staining in many tissues, including high staining in appendix and colon glandular cells (HPA tissue IHC). Its membrane association and perinuclear endosome localization are consistent with that distribution; NUMB has no transmembrane segment (UniProt P49757). HPA rates tissue IHC as Approved, with medium agreement between staining and RNA data and external verification pending (HPA tissue IHC).
| Membranous and cytoplasmic signal in appendix or colon glandular cells. | This fits the reported high staining in those cells and the broad tissue pattern (HPA tissue IHC). Assess cell boundaries and cytoplasm separately when scoring; a perinuclear component can also fit NUMB's endosome localization (UniProt P49757). |
| Predominantly nuclear staining, with little membranous or cytoplasmic signal. | A nuclear-only pattern conflicts with the reported tissue distribution and localization (HPA tissue IHC; UniProt P49757). Suspect an artefact or off-target staining, then compare a known-positive section and the negative detection control before interpreting it as NUMB. |
| Strong signal confined to adipocytes or lymph-node germinal center cells. | HPA reports NUMB as not detected in those specific cell populations (HPA tissue IHC). Check whether staining persists when the primary antibody is omitted; unexpected cell-restricted signal can reflect cross-reactivity or endogenous detection activity (general IHC practice). |
| Diffuse color across tissue, stroma and empty spaces. | A field-wide deposit lacks the cell and compartment pattern reported for NUMB (HPA tissue IHC). Check blocking, washes, detection reagent and chromogen development on a control section; diffuse background cannot establish cellular NUMB expression (general IHC practice). |
| No signal in appendix or colon glandular cells. | These are reported high-staining cell populations, so an absent signal makes the run difficult to interpret (HPA tissue IHC). Check section quality, retrieval and detection with appropriate controls before concluding that the specimen lacks NUMB (general IHC practice). |
💡Expected NUMB appearanceA convincing positive shows high membranous and cytoplasmic staining in appendix or colon glandular cells (HPA tissue IHC); nuclear-only or field-wide color is suspect against that reference pattern (HPA tissue IHC; general IHC practice).
| Cellular compartment | HPA describes membranous and cytoplasmic tissue staining (HPA tissue IHC). UniProt places NUMB at cell and endosome membranes, including perinuclear endosomes, despite no transmembrane segment (UniProt P49757). Score the visible compartment rather than treating all cellular color alike. |
| Tissue and cell selection | Appendix and colon glandular cells are reported high, whereas adipocytes and lymph-node germinal center cells are reported not detected (HPA tissue IHC). Use these as pattern references; HPA's medium staining–RNA agreement and pending external verification limit how definitive any single section can be (HPA tissue IHC). |
| Isoforms and epitope coverage | UniProt lists 9 NUMB isoforms and a PID domain at residues 33–193 (UniProt P49757). The supplied sources do not map this assay's epitope or establish which isoforms it detects; check antibody documentation before comparing isoform-dependent staining. |
| Antibody validation and detection | HPA lists CAB011478 tissue IHC as Approved, while HPA002874 has no tissue-IHC status in the supplied record (HPA antibodies). Validation belongs to the tested antibody; background from endogenous enzyme activity or detection reagents must be assessed with controls (general IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive glandular cells are blank. | The run may have failed; appendix and colon glandular cells are reported high (HPA tissue IHC). | Review tissue integrity, the antibody's IHC-P instructions, retrieval, dilution and detection; repeat with a positive control (general IHC practice). The supplied sources specify no retrieval condition or dilution. |
| Only nuclei stain strongly. | Nuclear-only staining disagrees with the membranous and cytoplasmic tissue pattern (HPA tissue IHC). | Compare with a positive tissue section and a no-primary control; review primary-antibody specificity and detection background before scoring (general IHC practice). |
| Adipocytes or germinal center cells stain strongly. | Both populations are reported not detected, although HPA validation remains pending external verification (HPA tissue IHC). | Check neighboring cell identity and a no-primary control. If signal persists without primary antibody, investigate endogenous detection activity; otherwise review antibody specificity (general IHC practice). |
| Brown deposit spreads beyond cell outlines. | Diffuse staining does not match HPA's cellular membrane and cytoplasm pattern (HPA tissue IHC); excess detection background is possible (general IHC practice). | Inspect the no-primary control, blocking and wash steps, then adjust detection and chromogen development according to reagent instructions (general IHC practice). |
| Positive and comparison tissues both look weak. | HPA reports different cell-level intensities, including high glandular staining and undetected adipocytes (HPA tissue IHC); weak color everywhere may indicate a technical issue (general IHC practice). | Verify the positive tissue and control staining first. Review retrieval and antibody concentration using the catalog antibody's IHC-P instructions; no NUMB-specific fixation sensitivity is reported in the supplied sources. |
| Does an IF/ICC junctional pattern validate this IHC-P result? | HPA calls IF/ICC junction localization uncertain, plasma membrane supported and cytosol uncertain; CAB011478 ICC is Uncertain (HPA subcellular; HPA antibodies). | Use the IF/ICC result as localization context only. Judge paraffin-section staining against tissue IHC cell patterns and the IHC validation for the antibody used (HPA tissue IHC; HPA antibodies). |
Experiment evidence
IHC & IF images showing NUMB staining
PB9301 · IHCIHC analysis of NUMB using anti-NUMB antibody (PB9301). NUMB was detected in a paraffin-embedded section of human liver cancer tissue. Heat
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.
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 its glandular cells (HPA: High in appendix glandular cells); use adipose tissue as the negative comparator and expect adipocytes to remain unstained (HPA: Not detected in adipocytes). On the appendix slide, compare glandular staining with any unstained neighboring cells and background, without assuming those cells are NUMB-negative (internal comparison: standard IHC practice).
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 rabbit IgG isotype control, and NUMB-knockout tissue or a validated peptide-block control (caption: rabbit primary antibody; controls: standard IHC practice). Check endogenous peroxidase activity in the appendix section before interpreting DAB signal (caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the PB9301 tissue-IHC caption does not state the fixative (caption: fixative not stated). The reported paraffin-section staining used heat retrieval in EDTA at pH 8.0; this supports that workflow but does not establish that retrieval is required for all specimens (caption: EDTA retrieval). The supplied evidence does not establish whether frozen sections or IF would be easier; check appendix background from endogenous peroxidase when scoring glandular DAB staining (caption: HRP/DAB detection; 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 → |
Section 3
Advanced NUMB IHC Tips
Troubleshoot NUMB staining by checking retrieval, compartment, cell type and controls before comparing signal across paraffin sections (UniProt P49757; HPA tissue IHC).
How should I retrieve NUMB in paraffin sections with PB9301?
Start with heat-mediated retrieval in EDTA at pH 8.0 for PB9301 paraffin-section IHC (datasheet PB9301). The reported liver cancer section was stained after that retrieval, followed by 10% goat-serum blocking and 2 μg/ml primary antibody overnight at 4°C (PB9301 tissue-IHC caption). If staining is weak, verify heating, cooling and buffer pH across the rack before testing another retrieval condition on matched sections (standard IHC practice). Compare changes within the same cell compartment, since NUMB has membranous and cytoplasmic tissue staining and retrieval can change apparent signal distribution (HPA tissue IHC; standard IHC practice).
Could fixation be causing weak or patchy NUMB staining?
The PB9301 image describes a paraffin-embedded liver cancer section but does not state its fixative, so NUMB-specific fixation sensitivity is unknown (PB9301 tissue-IHC caption). Record the fixative and fixation duration for each specimen, then compare similarly processed sections with the same pH 8.0 EDTA retrieval (standard IHC practice; datasheet PB9301). If staining differs between batches, check section thickness, deparaffinisation and retrieval uniformity before assigning the difference to NUMB abundance (standard IHC practice). Use a matched positive-control section in each run; do not infer fixation tolerance from NUMB’s membrane association, phosphorylation sites or tissue profile (UniProt P49757; HPA tissue IHC).
Where should NUMB staining appear within a positive cell?
Expect membranous and cytoplasmic staining in many tissues, while assessing each cell population separately (HPA tissue IHC). NUMB is associated with cell and endosome membranes and can localise to perinuclear endosomes in an AAK1-dependent manner, although it has no transmembrane segment (UniProt P49757). Cell-junction staining in the HPA subcellular record is uncertain; plasma-membrane localisation is supported, so treat a junction-only pattern cautiously (HPA subcellular). If a section shows exclusively nuclear or diffuse extracellular DAB, compare morphology, a negative control and a positive-control section before calling it NUMB (UniProt P49757; standard IHC practice).
Could NUMB isoforms or modifications explain inconsistent staining?
NUMB has 9 annotated isoforms, so an antibody’s epitope may determine which forms contribute to tissue staining (UniProt P49757; standard IHC practice). Its PID domain spans residues 33–193, and annotated phosphorylation includes threonine 102 and serines 194 and 244 (UniProt P49757). Check the catalog antibody’s stated immunogen or epitope against the isoforms under study; the supplied PB9301 caption does not identify that epitope (PB9301 tissue-IHC caption). When patterns disagree between antibodies, compare their epitope coverage and matched-section staining before interpreting the difference as altered NUMB expression (standard IHC practice).
How can IF help assess NUMB localisation alongside a cell-type marker?
For a separate IF experiment, multiplex NUMB with a marker identifying the glandular cells being assessed, since HPA reports high NUMB staining in appendix glandular cells (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores and place the weaker signal in a channel with less tissue autofluorescence; inspect unstained tissue in those channels first (standard IF practice). NUMB lacks a transmembrane segment and associates with membranes, so use gentle permeabilisation when the antibody epitope is cytosol-facing or intracellular (UniProt P49757; standard IF practice). Check single-label controls for bleed-through and compare membrane, junctional and perinuclear signal without treating the IHC caption as an IF fixation protocol (HPA subcellular; PB9301 tissue-IHC caption; standard IF practice).
What should I check when NUMB DAB staining looks diffuse?
First examine a section without primary antibody for secondary-reagent background and incomplete endogenous-peroxidase blocking (standard chromogenic IHC practice). The PB9301 image used 10% goat-serum blocking, 2 μg/ml primary antibody overnight at 4°C, and an HRP–DAB detection workflow (PB9301 tissue-IHC caption). If the control is clean, compare shorter primary exposure or lower primary concentration on adjacent sections while preserving the same pH 8.0 EDTA retrieval (datasheet PB9301; standard IHC practice). Evaluate cytoplasmic and membranous staining against cell outlines; pooled brown haze across stroma or section edges should not be scored as NUMB-positive cells (HPA tissue IHC; standard IHC practice).
How should I score NUMB staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because NUMB staining is reported as membranous and cytoplasmic in most tissues (HPA tissue IHC). For chromogenic IHC, record the percentage of positive cells and intensity by category, then calculate an H-score from the category percentages if intensity matters (standard IHC practice). Normalise positive-cell counts to the number of eligible cells, or cell density to the measured tissue area in mm²; exclude folds, necrosis and missing tissue (standard IHC practice). Use identical thresholds, exposure settings for captured images and compartment rules across batches, with a reference section to monitor staining drift (standard IHC practice).
How do I distinguish genuine NUMB staining from artefact?
Call staining convincing when it follows cell boundaries or cytoplasm in intact cells and recurs in comparable sections (HPA tissue IHC; standard IHC practice). HPA reports high staining in appendix glandular cells but no detection in adipocytes, providing cell-population references rather than universal positive and negative controls (HPA tissue IHC). Be cautious with isolated nuclear staining, necrotic areas, tissue edges or widespread DAB in a no-primary control; those patterns warrant review of morphology and endogenous-peroxidase blocking (UniProt P49757; standard IHC practice). HPA rates the tissue profile Approved with medium RNA–protein consistency and pending external verification, so corroborate unexpected biological claims independently (HPA tissue IHC).
Boster reagents
Best NUMB / Protein numb homolog IHC Antibodies
Two anti-NUMB antibodies have human tissue IHC images (catalog image captions); one also has a human lung IF image (A01206 image caption). Catalog reactivity spans human, monkey, mouse and rat (catalog applications/reactivity).
Real IHC data
Anti-NUMB Antibody ®
Cat # PB9301
Real IHC data
Anti-Protein numb homolog NUMB Antibody
Cat # A01206
PB9301 shows IHC in paraffin sections of human liver cancer and lung cancer tissue (PB9301 IHC image captions). A01206 shows IHC and IF in human lung tissue (A01206 image captions).
Which to pick: For paraffin-section IHC, choose PB9301 if its documented retrieval and detection conditions are useful: EDTA pH 8.0 retrieval and DAB detection are shown in its own caption (PB9301 IHC image caption); the fixative is unreported (PB9301 IHC image caption). For tissue IF, choose A01206, which has a human lung IF image and lists IF as an application; ICC validation and the tissue fixative are unreported (A01206 IF image caption; A01206 applications). For cross-species planning, PB9301 lists human, monkey, mouse and rat reactivity, while A01206 lists human, mouse and rat; the cited IHC images show human tissue only, and clonality is unreported for both (catalog reactivity; catalog IHC image captions; catalog clone fields).
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.
Immunohistochemistry protocol sheet for NUMB (Protein numb homolog). Expected localisation Membranous and cytoplasmic staining in most tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PB9301). Validated IHC antibody: PB9301 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet PB9301); 2. Fixation: Image fixative and duration unreported (datasheet PB9301); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: EDTA pH 8.0 (datasheet PB9301); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9301); 8. Primary antibody: Rabbit anti-NUMB, 2-5μg/ml (datasheet PB9301); 9. Primary incubation: Overnight at 4 °C (datasheet PB9301). Watch for: Fixation: Keep fixation conditions consistent across sections (standard IHC practice; not target-specific); Caveat: IHC staining has medium concordance with RNA data (HPA tissue IHC); Isoform / epitope: 9 isoforms; check epitope conservation across forms (UniProt); Regulation: Staining-linked expression regulation is unreported (UniProt). Evidence: UniProt P49757, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the cell junctions. In addition localized to the…, Datasheet PB9301, PMC5354663, PMC12035580. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NUMB/immunohistochemistry-ihc-guide.