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CUBN / Cubilin · IHC design guide
Design Immunohistochemistry for CUBN
Plan paraffin-section CUBN IHC using kidney tubules as a positive reference and cytoplasmic and membranous staining as the expected tissue pattern (HPA tissue IHC). Keep fixation consistent and optimise the catalog antibody within its 1:50–1:200 IHC dilution range (datasheet).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CUBN 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 and membranous tissue staining (HPA tissue IHC) | |
| Staining pattern | Proximal renal tubules and small intestinal epithelia: cytoplasmic/membranous (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Kidney | |
| Negative control | Adipose tissue+4 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 | Plasma-membrane localisation depends on AMN interaction (UniProt) | |
| Regulation | Kidney-enriched RNA expression (HPA tissue RNA) | |
| Isoform / epitope | No isoforms listed; mature chain aa 36–3623; no transmembrane segment (UniProt) |
Section 1
Recommended CUBN IHC & IF Protocols
The catalog antibody protocol is followed by published CUBN IHC methods for renal tissue (PMC6247592) and human renal cell carcinoma tissue microarrays (PMC5215231).
| Sample | Paraffin-embedded human kidney tissue; fixative not specified (datasheet M03670) |
| Fixation | Image fixative and duration unreported (datasheet M03670); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit monoclonal (clone GA-3) anti-CUBN, 1:50-1:200 (datasheet M03670) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CUBN-positive staining in cells in tubules of kidney (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and membranous expression in the proximal renal tubules and small intestine epithelia. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page antigen retrieval); the published excerpts do not specify retrieval (PMC6247592; PMC5215231).
Section 2
What Is the Expected CUBN Staining Pattern?
CUBN should stain proximal renal tubule cells and small intestinal epithelium with an apical membranous and cytoplasmic pattern (HPA: tissue IHC; UniProt O60494: apical membrane, endosomes). Kidney tubular staining is High, and HPA rates its tissue IHC profile Enhanced for consistency with RNA expression (HPA: kidney; HPA: reliability). CUBN lacks a transmembrane segment and depends on AMN for plasma membrane localisation (UniProt O60494: topology and subunit).
| Strong staining outlines the luminal edge of renal tubule cells, with some cytoplasmic staining. | This fits the expected renal pattern: HPA reports High staining in kidney tubule cells and describes cytoplasmic and membranous expression in proximal tubules (HPA: tissue IHC). An apical emphasis is consistent with CUBN localisation (UniProt O60494: subcellular location). Judge the pattern within tubules, since the supplied HPA level applies to tubular cells (HPA: kidney). |
| Small intestinal epithelial cells show membranous and cytoplasmic staining. | This is an expected tissue and cell type (HPA: tissue IHC; UniProt O60494: tissue specificity). The supplied HPA profile does not assign these cells a High staining level, so compare their compartment pattern without requiring renal intensity (HPA: tissue IHC). |
| Staining is predominantly nuclear or distributed uniformly across unrelated compartments. | That differs from HPA's cytoplasmic and membranous tissue pattern and UniProt's membrane and endocytic locations (HPA: tissue IHC; UniProt O60494: subcellular location). Check morphology and controls before scoring it as CUBN; nonspecific detection or interpretation of counterstain is possible (general IHC practice). |
| Strong staining appears in adipocytes or adrenal glandular cells. | These cell types are listed as Not detected in the supplied tissue IHC data (HPA: adipose tissue and adrenal gland). Treat the result as discordant and assess cross-reactivity and endogenous detection activity with appropriate controls (general IHC practice). A discordant stain alone does not establish either cause. |
| Kidney tubules show no staining despite a visible detection reaction elsewhere. | The result conflicts with High kidney tubular staining in HPA's reference profile (HPA: kidney). Review section quality, tissue identity and morphology, then check antibody and detection controls (general IHC practice). A failed positive control limits interpretation of negative study sections (general IHC practice). |
💡Expected CUBN appearanceCall a positive result when renal proximal tubule cells show strong apical membranous staining with cytoplasmic signal (HPA: High kidney tubular staining and tissue pattern; UniProt O60494: apical location); isolated nuclear staining or strong staining in HPA Not detected cell types is discordant (HPA: tissue IHC).
| Tissue and cell selection | Kidney proximal tubules provide a supported positive reference: HPA reports High tubular staining, and UniProt reports CUBN in kidney cortex at protein level (HPA: kidney; UniProt O60494: tissue specificity). Small intestinal epithelium is another expected site, while the listed adipocytes and adrenal glandular cells are Not detected comparators (HPA: tissue IHC). |
| Apical organisation and AMN | CUBN has no transmembrane segment and depends on AMN for plasma membrane localisation (UniProt O60494: topology and subunit). Interpret an apical rim in the context of epithelial polarity and morphology (UniProt O60494: apical cell membrane; general IHC practice). |
| Intracellular signal | Cytoplasmic staining can fit the tissue profile (HPA: tissue IHC). UniProt also places CUBN in coated pits, endosomes and lysosome membranes, supporting intracellular signal alongside membrane staining; those annotations do not identify individual puncta on a chromogenic section (UniProt O60494: subcellular location). |
| Processing and antibody epitope | UniProt annotates a signal peptide at residues 1–23, a propeptide at 24–35 and a mature chain at 36–3623 (UniProt O60494: processing). No epitope location is supplied for the staining antibody, so these annotations cannot predict which processed form it detects or explain a missing tissue signal. |
| IHC validation | Both listed antibodies, HPA004133 and HPA043854, have Enhanced IHC status, and HPA rates the tissue profile Enhanced for agreement with RNA expression (HPA: antibodies; HPA: reliability). This supports the reference distribution but does not establish that every antibody or detection run will reproduce it. |
| IF/ICC applicability? | HPA supplies no CUBN ICC-IF images or assigned main subcellular location (HPA: subcellular). Use the IHC tissue pattern and UniProt localisation as expectations, while treating IF/ICC staining performance as unverified by the supplied HPA record (HPA: tissue IHC; UniProt O60494: subcellular location). |
| Situation | Likely cause | Next action |
|---|---|---|
| No staining in the kidney positive reference | The run or tissue may be unsuitable; a CUBN-specific failure mechanism cannot be assigned from these records (HPA: High kidney tubular staining). | Verify proximal tubule morphology and section integrity, then review antibody, detection and control performance before scoring other sections negative (general IHC practice). |
| Tubules stain, but the luminal edge is hard to distinguish | Cytoplasmic staining is part of the reported pattern, and section orientation can obscure epithelial polarity (HPA: tissue IHC; general IHC practice). | Examine well-oriented tubules and compare the luminal rim with cytoplasm; record both compartments instead of calling all tubular colour apical (general IHC practice). |
| Colour spreads broadly across tissue or obscures cell borders | Diffuse signal may reflect detection background; the reference pattern is cell and compartment specific (general IHC practice; HPA: tissue IHC). | Inspect the negative reagent control, blocking and wash steps, then adjust detection conditions according to the assay instructions (general IHC practice). |
| Unexpected staining appears in HPA Not detected cells | Cross-reactivity or endogenous chromogenic detection activity may contribute; the image alone cannot distinguish them (HPA: listed negative cell types; general IHC practice). | Compare a negative reagent control and an appropriate endogenous activity control, then recheck tissue and cell identity (general IHC practice). |
| Signal is mainly nuclear | Nuclear localisation is outside the supplied HPA tissue pattern and UniProt location list (HPA: tissue IHC; UniProt O60494: subcellular location). | Check counterstain appearance and controls, then score CUBN only where cell type and compartment agree with the reference pattern (general IHC practice; HPA: tissue IHC). |
| Signal remains weak after a run with working controls | The supplied sources do not establish CUBN-specific antigen retrieval or fixation sensitivity; weak signal has several possible assay causes. | Review tissue preservation and the antibody's validated IHC conditions; if changing antigen retrieval, compare conditions with the kidney positive reference in the same run (general IHC practice; HPA: High kidney tubular staining). |
Experiment evidence
IHC & IF images showing CUBN staining
M03670 · IHCImmunohistochemical analysis of paraffin-embedded human kidney, using Cubilin Antibody.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000107611-CUBN/tissue/cerebral+cortex#imid_18610884
HPA · Small intestineHuman Protein Atlas IHC, Small intestine. Source: https://www.proteinatlas.org/ENSG00000107611-CUBN/tissue/small+intestine#imid_18611377
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000107611-CUBN/tissue/colon#imid_18611394
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000107611-CUBN/tissue/liver#imid_18610819
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CUBN IHC & IF
🧪Run kidney first: cells in tubules should stain (High; HPA: kidney, cells in tubules). Use adipose tissue adipocytes as the negative tissue (HPA: adipocytes, Not detected); on the kidney slide, glomerular and interstitial cells should lack the distinct tubular staining pattern and serve as background comparators, without assuming they are CUBN-free (UniProt O60494: proximal tubule expression).
Positive control tissue: Kidney (Cells in tubules, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for CUBN; derive a cell-line control from the positive tissue's cell type (Cells in tubules) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary (secondary-only) slide, an immunoglobulin control matched to the primary antibody’s host species and isotype, and a CUBN knockout sample or a matching immunizing-peptide competition control if available (standard IHC controls). For kidney chromogenic IHC, quench endogenous peroxidase and block endogenous biotin if using avidin–biotin detection (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and retrieval dependency are unreported in the supplied evidence; optimize retrieval using matched positive and negative sections (supplied evidence; standard IHC practice). The M03670 caption shows staining in paraffin-embedded human kidney but does not report a fixative, so fixation cannot be assumed (selected-SKU tissue-IHC caption). Paraffin IHC has the supplied tissue example, while frozen sections and IF are not established as easier here; kidney endogenous peroxidase or biotin can complicate chromogenic interpretation unless controlled (selected-SKU tissue-IHC caption; standard IHC practice). The selected M03670 tissue-IHC caption documents paraffin sections, but does not specify the fixative (selected-SKU IHC image M03670).
HPA tissue IHC evidence for CUBN
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High consistency between antibody staining and RNA expression data.). 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 |
|---|---|---|---|---|
| Kidney | Cells in tubules | High | 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 | Endocrine 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 CUBN IHC Tips
Troubleshoot CUBN staining by checking retrieval, compartment, epithelial cell identity and controls before interpreting changes in chromogenic signal.
How should I retrieve CUBN in paraffin sections when staining is weak?
Use citrate buffer at pH 6.0 for heat induced retrieval at 95–98 °C for 20 min (page retrieval rule). Run a known positive kidney section alongside the test section and keep heating and cooling conditions consistent across slides (HPA: high staining in kidney tubule cells; standard IHC practice). If staining remains weak, optimize heating time on adjacent sections before testing an alternative retrieval buffer as a fallback (standard IHC practice). Compare apical and cytoplasmic staining in tubules, because CUBN occupies the apical membrane and endocytic compartments (UniProt O60494 localisation; HPA: proximal renal tubule pattern).
Could fixation explain loss of CUBN staining in my kidney sections?
Target specific fixation sensitivity is unknown from the supplied evidence; the selected M03670 image shows paraffin embedded human kidney but does not state its fixative (caption M03670). Record your fixative, fixation duration and tissue thickness before comparing staining across specimens (standard IHC practice). If signal varies, stain sections processed with different fixation durations in the same run, using the same retrieval and detection conditions (standard IHC practice). Include kidney tubules as a positive control and judge both signal and morphology; neither the HPA tissue pattern nor CUBN topology establishes a fixation effect (HPA: kidney tubule staining; UniProt O60494 topology).
Should CUBN stain the apical border, cytoplasm, or both?
Expect apical membrane staining with possible intracellular puncta in absorptive epithelia: CUBN is assigned to the apical membrane, coated pits, endosomes and lysosome membrane (UniProt O60494 localisation). HPA describes cytoplasmic and membranous staining in proximal renal tubules and small intestine epithelia (HPA: tissue IHC profile). Examine intact epithelial profiles at high magnification, separating luminal border signal from diffuse cytoplasmic chromogen and luminal debris (standard IHC practice). CUBN lacks a transmembrane segment and depends on AMN for plasma membrane location, so an exclusively diffuse pattern warrants comparison with morphology and controls before interpretation (UniProt O60494 topology and AMN dependence; standard IHC practice).
How can epitope placement change my CUBN IHC result?
Check the antibody's stated immunogen or mapped epitope before interpreting a negative section, because the supplied record identifies 0 isoforms and does not locate this antibody's epitope (UniProt O60494 isoform record; caption M03670). Mature CUBN follows a signal peptide at residues 1–23 and a propeptide at 24–35 (UniProt O60494 processing). The protein contains numerous CUB domains and 47 listed glycosylation sites, which make epitope identity relevant when comparing antibodies (UniProt O60494 domains and glycosylation). Test any second antibody on matched positive and negative sections, and report its epitope information if available (standard IHC practice).
How should I adapt the CUBN localisation check for multiplex IF?
Use a renal proximal tubule cell marker in a separate channel to identify the expected positive epithelium, then assess whether CUBN follows the luminal border or intracellular vesicles (UniProt O60494 tissue specificity and localisation; standard IF practice). Choose fluorophores after imaging unstained tissue, favoring channels with less tissue autofluorescence and checking each channel separately (standard IF practice). For an epitope accessible at the tissue surface, begin with mild or no permeabilisation; for an intracellular epitope, compare controlled permeabilisation conditions (standard IF practice). The selected M03670 caption documents paraffin section IHC, while the supplied HPA subcellular record provides no ICC/IF images to validate an IF pattern (caption M03670; HPA: subcellular record).
What should I check when chromogen obscures CUBN in renal tubules?
First inspect a no primary control and a known negative tissue section to separate detection background from candidate CUBN staining (standard IHC practice; HPA: adipocytes in adipose tissue not detected). In peroxidase IHC, apply an endogenous peroxidase block before chromogen development and compare it with an otherwise matched slide (standard IHC practice). Titrate primary antibody and detection incubation while preserving a positive kidney tubule control; no dilution is supplied for this selected antibody (standard IHC practice; HPA: high in kidney tubule cells; caption M03670). Treat staining outside epithelial profiles or concentrated at torn edges as suspect until controls and morphology support it (standard IHC practice).
How should I score CUBN staining across kidney specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define proximal tubule epithelial profiles before scoring, because CUBN is reported in those cells and HPA rates kidney tubule staining high (UniProt O60494 tissue specificity; HPA: kidney tubule cells, High). On matched chromogenic sections, record either an H score from intensity and percent positive cells or the percent positive epithelial cells, using one prespecified method throughout (standard IHC practice). Normalize the result to the number or area of evaluable proximal tubules, rather than total section area, and exclude folds and damaged regions (standard IHC practice). Score apical and cytoplasmic signal separately when distribution matters, since both patterns are reported (UniProt O60494 localisation; HPA: tissue IHC profile).
Which patterns support a true CUBN signal rather than an artefact?
A credible result follows epithelial morphology in kidney proximal tubules, with apical and sometimes cytoplasmic staining, and agrees with a positive control (UniProt O60494 tissue specificity and localisation; HPA: tissue IHC profile; standard IHC practice). Diffuse nuclear staining is discordant with the supplied membrane and endocytic localisation, so review it against a no primary control (UniProt O60494 localisation; standard IHC practice). Staining limited to section edges, folds or necrotic regions requires caution because tissue damage can trap detection reagents (standard IHC practice). If a peroxidase reaction persists without primary antibody, investigate endogenous enzyme activity before calling the chromogen CUBN positive (standard IHC practice).
Boster reagents
Best CUBN / Cubilin IHC Antibodies
M03670 has real IHC data from paraffin-embedded human kidney (M03670 IHC image caption) and lists human reactivity (catalog reactivity). No IF/ICC data are supplied (catalog applications and IF image alts).
Real IHC data
Anti-Cubilin CUBN Rabbit Monoclonal Antibody
Cat # M03670
M03670 is listed for IHC with human reactivity (catalog applications and reactivity). Its own IHC figure shows paraffin-embedded human kidney stained for Cubilin (M03670 IHC image caption).
Which to pick: For tissue IHC, choose M03670: it is a rabbit monoclonal (catalog host and clone) with an IHC image from paraffin-embedded human kidney (M03670 IHC image caption); the fixative is unreported (M03670 IHC image caption). There is no supported IF/ICC pick (catalog applications and IF image alts). There is no supported cross-species pick because M03670 lists human reactivity only (catalog reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O60494 (CUBN_HUMAN, Cubilin).
- Human Protein Atlas. CUBN tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CUBN subcellular location (ICC-IF): Highest expression in ASC52telo: 2.8 nTPM.
- Human Protein Atlas. CUBN antibody validation summary (2 antibodies).
- New Mutation of Coenzyme Q(10) Monooxygenase 6 Causing Podocyte Injury in a Focal Segmental Glomerulosclerosis Patient. Chinese medical journal 2018 — PMC6247592.
- A systematic search strategy identifies cubilin as independent prognostic marker for renal cell carcinoma. BMC cancer 2017 — PMC5215231.
- Cubam receptor-mediated endocytosis in hindgut-derived pseudoplacenta of a viviparous teleost (Xenotoca eiseni). The Journal of experimental biology 2021 — PMC8278012.
- Distinct intestinal adaptation for vitamin B12 and bile acid absorption revealed in a new mouse model of massive ileocecal resection. Biology open 2017 — PMC5612230.
- PubMed PMID:9572993 — UniProt-cited evidence.
- PubMed PMID:15164054 — UniProt-cited evidence.
- PubMed PMID:10371504 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CUBN (Cubilin). Expected localisation Cytoplasmic and membranous tissue staining (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: M03670 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Kidney (Cells in tubules); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human kidney tissue; fixative not specified (datasheet M03670); 2. Fixation: Image fixative and duration unreported (datasheet M03670); 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-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit monoclonal (clone GA-3) anti-CUBN, 1:50-1:200 (datasheet M03670); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Plasma-membrane localisation depends on AMN interaction (UniProt); Isoform / epitope: No isoforms listed; mature chain aa 36–3623 ; no transmembrane segment (UniProt); Regulation: Kidney-enriched RNA expression (HPA tissue RNA). Evidence: UniProt O60494, HPA tissue IHC · Enhanced, HPA cell-line RNA · Highest expression in ASC52telo: 2.8 nTPM, Datasheet M03670, PMC6247592, PMC5215231. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CUBN/immunohistochemistry-ihc-guide.