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ITGB5 / Integrin beta-5 · IHC design guide
Design Immunohistochemistry for ITGB5
Plan chromogenic ITGB5 IHC on paraffin sections using an IHC-validated antibody (datasheet M04201). Assess cytoplasmic staining by cell type while keeping its annotated membrane location in view (HPA tissue IHC; UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ITGB5 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 tissue staining (HPA tissue IHC); cell membrane location (UniProt) | |
| Staining pattern | General cytoplasmic staining across sampled tissues (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet M04201) | |
| Positive control | Breast+4 more · see all | |
| Negative control | Bone marrow+3 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 | Lung macrophage staining can complicate cell attribution (HPA tissue IHC) | |
| Regulation | Expression regulation is not annotated (UniProt) | |
| Isoform / epitope | No isoforms annotated; extracellular and cytoplasmic regions differ (UniProt) |
Section 1
Recommended ITGB5 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet M04201) is accompanied by published ITGB5 staining protocols for HCC, tongue squamous cell carcinoma, and colorectal cancer (PMC13570297; PMC11344732; PMC11914080).
| Sample | Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet M04201) |
| Fixation | Image fixative and duration unreported (datasheet M04201); 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 M04201); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M04201) |
| Primary antibody | Mouse monoclonal (clone 9E2) anti-ITGB5, 2μg/ml (datasheet M04201) |
| Primary incubation | Overnight at 4 °C (datasheet M04201) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet M04201) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ITGB5-positive staining in glandular cells of breast (HPA tissue IHC: Medium). HPA tissue profile: General cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet M04201). The tongue carcinoma protocol specifies microwave retrieval but gives no buffer (PMC11344732).
Section 2
What Is the Expected ITGB5 Staining Pattern?
ITGB5 is a cell membrane protein with an extracellular region and one transmembrane segment (UniProt P18084 topology). In paraffin-section IHC, HPA reports general cytoplasmic staining, including medium staining in lung macrophages, cerebral-cortex endothelial cells, and breast glandular cells (HPA: tissue IHC; reliability Enhanced, with medium consistency between staining and RNA data). Interpret the observed cytoplasmic pattern alongside its membrane location (HPA: tissue IHC; UniProt P18084 subcellular location).
| Lung macrophages or cerebral-cortex endothelial cells show medium chromogenic staining (HPA: tissue IHC). | This matches observed positive cells; record whether staining is cytoplasmic or outlines the cell, since HPA reports general cytoplasmic IHC while UniProt places ITGB5 at the membrane (HPA: tissue IHC; UniProt P18084 subcellular location). |
| Staining is confined to nuclei, with no corresponding cytoplasmic or cell-edge signal. | An isolated nuclear pattern does not match the reported IHC profile or membrane location (HPA: tissue IHC; UniProt P18084 subcellular location). Check counterstain and detection controls before assigning it to ITGB5 (general IHC practice). |
| Bone-marrow hematopoietic cells stain strongly while the expected positive cells stain weakly. | HPA lists those hematopoietic cells as not detected (HPA: bone marrow). Investigate nonspecific antibody binding or endogenous detection activity using reagent controls (general IHC practice); the pattern alone cannot distinguish them. |
| Color covers tissue broadly, including spaces or structures without a clear cell-specific pattern. | Diffuse deposit is difficult to score as ITGB5; uneven reagent coverage, inadequate washing, or detection background can produce it (general IHC practice). Compare the affected area with a no-primary control and intact positive cells (general IHC practice). |
| No staining appears in lung macrophages or breast glandular cells. | Both are listed at medium staining (HPA: lung; HPA: breast). A blank run calls for checks of tissue preservation, retrieval, primary-antibody setup, and detection using a known-positive section (general IHC practice); it does not by itself establish absent ITGB5. |
💡Expected ITGB5 appearanceA credible positive shows medium cell-associated staining in HPA-positive cells, such as lung macrophages or cerebral-cortex endothelial cells (HPA: tissue IHC), with cytoplasmic staining compatible with HPA IHC and possible cell-edge emphasis compatible with membrane topology (HPA: tissue IHC; UniProt P18084 topology); isolated nuclear color or broad cell-free deposit is suspect (general IHC interpretation).
| Cellular location and epitope orientation | ITGB5 spans the membrane at residues 720–742, with residues 24–719 extracellular and 743–799 cytoplasmic (UniProt P18084 topology). Antibody epitope location is not supplied, so these boundaries cannot predict which staining condition will expose it. |
| Observed tissue context | HPA reports medium staining in kidney glomerular cells, liver cholangiocytes, and pancreatic endocrine cells, but no detection in esophageal squamous epithelium (HPA: tissue IHC). Judge signal in its identified cell population, not from whole-section color alone (general IHC practice). |
| Evidence behind the IHC pattern | HPA rates tissue IHC reliability Enhanced while describing medium staining-to-RNA consistency; HPA001820 is IHC Enhanced and CAB022050 IHC Approved (HPA: tissue IHC; HPA: antibody validation). These ratings support comparison with the observed pattern, not a guarantee for every section. |
| IF/ICC Q: Where should ITGB5 appear? | A: HPA approves plasma-membrane localisation and labels mitochondrial localisation uncertain; images are listed for U-251MG and U2OS (HPA: subcellular ICC-IF). Treat a mitochondrial-only result cautiously. This IF/ICC observation does not set a paraffin-IHC staining condition. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive cells are blank, and the no-primary control is clean. | A missed primary-antibody step, unsuitable dilution, or underdeveloped detection can cause a blank IHC run (general IHC practice); target-specific retrieval sensitivity is unreported in the supplied evidence. | Confirm primary-antibody application and the IHC-P setup, then check retrieval and detection with a known-positive section (general IHC practice). Use HPA-positive cells, such as lung macrophages, as the pattern reference (HPA: lung). |
| Signal is weak in every expected cell population. | Weak detection or an unsuitable antibody concentration can reduce chromogenic contrast generally (general IHC practice). The supplied UniProt and HPA records give no ITGB5-specific dilution or retrieval setting. | Compare positive and no-primary controls, verify detection reagents, and optimize the catalog antibody's IHC-P conditions within its stated instructions (general IHC practice). Score cell populations only after controls behave as expected. |
| Strong color appears in HPA-negative epithelium. | Esophageal squamous and nasopharyngeal respiratory epithelial cells are listed as not detected (HPA: tissue IHC). Nonspecific primary binding or endogenous detection activity could explain an unexpected signal (general IHC practice). | Compare a no-primary control and the expected positive cells on a matched run; inspect whether the color follows tissue boundaries or a plausible cell pattern (general IHC practice). Avoid scoring unexpected color as ITGB5 without corroboration. |
| Only nuclei stain in an otherwise clear section. | Nuclear-only staining conflicts with the membrane assignment and HPA's general cytoplasmic IHC profile (UniProt P18084 subcellular location; HPA: tissue IHC). Counterstain or detection background may be mistaken for target color (general IHC practice). | Review the chromogen and counterstain separately, check no-primary controls, and repeat the comparison in an HPA-positive cell population (general IHC practice; HPA: tissue IHC). |
| Background obscures cell boundaries across the section. | Incomplete washing, excess detection reagent, or nonspecific antibody binding can obscure localisation in chromogenic IHC (general IHC practice); this appearance does not establish ITGB5 abundance. | Check the no-primary control, washing and blocking steps, then adjust the general detection setup before rescoring cytoplasmic or cell-edge signal (general IHC practice; HPA: tissue IHC; UniProt P18084 topology). |
| A section stains differently from the HPA example despite working controls. | HPA reports medium consistency between antibody staining and RNA data, and its tissue entries describe specified cell populations (HPA: tissue IHC). A different sampled cell mix can change the apparent whole-section result (general IHC interpretation). | Identify and score the relevant cells separately; compare an HPA-listed positive population and a listed not-detected population in the same run when available (HPA: tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing ITGB5 staining
M04201 · IHCIHC analysis of Integrin beta 5/ITGB5 using anti-Integrin beta 5/ITGB5 antibody (M04201). Integrin beta 5/ITGB5 was detected in paraffin-emb
M04201 · IHCIHC analysis of Integrin beta 5/ITGB5 using anti-Integrin beta 5/ITGB5 antibody (M04201). Integrin beta 5/ITGB5 was detected in paraffin-emb
A04201-1 · IHCIHC analysis of ITGB5 using anti-ITGB5 antibody (A04201-1). ITGB5 was detected in paraffin-embedded section of human rectal cancer tissue. H
A04201-1 · IFIF analysis of Integrin beta 5/ITGB5 using anti-Integrin beta 5/ITGB5 antibody (A04201-1). Integrin beta 5/ITGB5 was detected in immunocytoc
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000082781-ITGB5/tissue/cerebral+cortex#imid_932384
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000082781-ITGB5/tissue/colon#imid_932302
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000082781-ITGB5/tissue/liver#imid_932303
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000082781-ITGB5/tissue/kidney#imid_932320
HPA · ICC-IF U-251MGHuman Protein Atlas immunofluorescence, U-251MG, antibody CAB022050; green = ITGB5, blue = nucleus, red = microtubules.
HPA · ICC-IF U-251MGHuman Protein Atlas immunofluorescence, U-251MG, antibody CAB022050; green = ITGB5, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ITGB5 IHC & IF
🧪Run breast first and assess staining in glandular cells (HPA: breast glandular cells, Medium). Use esophagus squamous epithelial cells as the negative tissue (HPA: Not detected); on the breast slide, any unstained non-glandular cells should show only background signal, but their ITGB5-negative status is unverified (HPA: breast glandular cells, Medium).
Positive control tissue: Breast (Glandular cells, HPA Medium)
Negative control tissue: Bone marrow (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ITGB5 in U-251MG, U2OS, with annotated localisation: Plasma membrane (approved), Mitochondria (uncertain) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a concentration-matched mouse IgG isotype control with the appropriate monoclonal or polyclonal format (M04201 caption: mouse primary; standard IHC control practice). Use an ITGB5 knockout specimen as a biological negative; for SABC/DAB detection, check endogenous peroxidase and biotin background on the breast slide (M04201 caption: SABC/DAB; standard IHC control practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected M04201 paraffin-section caption does not state the fixative (M04201 caption). The caption uses heat-mediated EDTA retrieval at pH 8.0, but does not establish that retrieval is required; the supplied evidence does not establish whether frozen sections or IF are easier (M04201 caption; HPA: ICC-IF images). With SABC/DAB detection, endogenous biotin or peroxidase could complicate interpretation of breast staining, so assess background with the reagent controls (M04201 caption: SABC/DAB; standard IHC practice).
HPA tissue IHC evidence for ITGB5
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium 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 |
|---|---|---|---|---|
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | Endothelial cells | Medium | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | Medium | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | Medium | Protein (IHC) | HPA → |
| Kidney | Cells in glomeruli | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Nasopharynx | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Vagina | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ITGB5 IHC Tips
Troubleshoot chromogenic ITGB5 IHC in paraffin sections by checking retrieval, staining compartment, controls, and cell-specific scoring.
What retrieval should I try first when ITGB5 staining is weak?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 (datasheet M04201). The documented paraffin-section example then used 2 µg/mL primary antibody overnight at 4°C, so keep those conditions consistent while assessing retrieval (caption M04201). Compare a retrieved section with a no-retrieval section and a no-primary control in the same run to distinguish improved signal from retrieval-associated background (standard IHC practice). If staining remains weak, optimize heating time on serial sections before considering another buffer as a fallback (standard IHC practice). Record morphology alongside signal, since excessive heating can damage sections and complicate scoring (standard IHC practice).
How should I troubleshoot fixation-related variation in ITGB5 IHC?
Target-specific fixation sensitivity is unknown: the M04201 tissue image describes a paraffin section but does not state its fixative (caption M04201). Record the fixative and fixation duration for each specimen, and compare serial sections processed with the same EDTA pH 8.0 retrieval and antibody conditions (datasheet M04201; standard IHC practice). Inspect tissue preservation before interpreting a weak result, because poor morphology makes cellular localisation difficult to judge (standard IHC practice). Include a concurrently processed positive control and a no-primary control when comparing batches (standard IHC practice). Do not infer an ITGB5-specific fixation effect from its membrane topology or tissue staining profile (UniProt P18084 topology; HPA tissue IHC).
Should ITGB5 appear on the membrane or in the cytoplasm?
Prioritize cell-border staining when evaluating ITGB5 because its annotated location is the cell membrane and its transmembrane segment spans residues 720–742 (UniProt P18084). Its extracellular region spans 24–719, whereas residues 743–799 face the cytoplasm, so epitope location matters when judging the pattern (UniProt P18084 topology). HPA describes general cytoplasmic staining in tissue IHC; compare that pattern with membrane staining and control sections rather than rejecting it solely by compartment (HPA tissue IHC; standard IHC practice). HPA also lists plasma membrane as approved and mitochondrial localisation as uncertain in subcellular imaging (HPA subcellular). Document which cells stain and whether borders remain discernible at higher magnification (standard IHC practice).
How can epitope location explain inconsistent ITGB5 staining?
Check the catalog antibody’s stated immunogen before interpreting discordant staining; the supplied evidence does not identify its epitope (catalog antibody evidence). UniProt lists no alternative ITGB5 isoforms and places the extracellular region at residues 24–719 and cytoplasmic region at 743–799 (UniProt P18084). The extracellular region includes 8 annotated glycosylation sites, while Ser770 is an annotated modified residue in the cytoplasmic region (UniProt P18084). These annotations identify possible epitope contexts, but they do not establish which modification affects this antibody’s staining (UniProt P18084; catalog antibody evidence). Compare the reported immunogen with the staining compartment and test any proposed explanation on matched control sections (standard IHC practice).
How should I investigate ITGB5 localisation by multiplex IF?
Use IF as a separate assay and first confirm that the chosen antibody works under its IF fixation conditions; the M04201 paraffin IHC caption does not establish IF performance (caption M04201; standard IF practice). For a relevant cell check, pair ITGB5 with a macrophage marker such as CD68 in lung, where HPA reports medium macrophage staining (HPA tissue IHC; standard IF practice). Choose well-separated fluorophores and assess an unstained tissue section, favoring a longer-wavelength channel if tissue autofluorescence obscures the signal (standard IF practice). If the epitope is extracellular, try minimal permeabilisation; if cytoplasmic, use controlled permeabilisation after fixation (UniProt P18084 topology; standard IF practice). Compare single-stain and no-primary controls before assigning colocalisation (standard IF practice).
How can I reduce diffuse or misleading DAB background?
Begin with a no-primary section to assess detection-system background before changing the ITGB5 antibody concentration (standard IHC practice). The documented M04201 workflow used 10% goat serum, biotinylated goat anti-mouse secondary for 30 minutes at 37°C, a streptavidin–biotin complex, and DAB (caption M04201). For this biotin-based workflow, check endogenous biotin with an appropriate control and add a blocking step if it contributes signal (caption M04201; standard IHC practice). Apply a peroxidase block and inspect the no-primary section for residual enzyme-associated DAB deposition (standard IHC practice). If background persists, titrate the primary around the documented 2 µg/mL condition while keeping retrieval and development consistent (caption M04201; standard IHC practice).
What is a reproducible way to score ITGB5 IHC? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, since ITGB5 is annotated at the cell membrane while HPA reports general cytoplasmic tissue staining (UniProt P18084; HPA tissue IHC). For a selected cell population, report the percentage of positive cells and an H-score from intensity categories 0–3, with a possible range of 0–300 (standard IHC practice). If counting positive cells in tissue regions, report density per mm² of evaluable tissue and exclude folds, necrosis, and missing section areas (standard IHC practice). Normalize comparisons to the same cell type, sampled area, staining batch, and exposure or scan settings (standard IHC practice). Record membrane and cytoplasmic scores separately when both patterns occur (standard IHC practice).
How do I distinguish true ITGB5 staining from artefact?
Look for reproducible staining in intact cells with discernible borders, consistent with ITGB5’s annotated membrane location (UniProt P18084; standard IHC practice). Use cell identity as a check: HPA reports medium staining in lung macrophages and cerebral-cortex endothelial cells, while its tissue profile also describes general cytoplasmic expression (HPA tissue IHC). Treat staining confined to section edges, folds, or necrotic areas as suspect and compare it with intact regions on the same slide (standard IHC practice). A no-primary control helps reveal endogenous enzyme or detection-system signal, especially when DAB is used (standard IHC practice). Confirm an unexpected compartment or cell population across independent sections before calling it ITGB5-positive (standard IHC practice).
Boster reagents
Best ITGB5 / Integrin beta-5 IHC Antibodies
IHC images show ITGB5 staining in human paraffin-embedded cancer sections (catalog IHC captions); IF data show staining in A431 cells (A04201-1 IF caption).
Real IHC data
Anti-Integrin beta 5/ITGB5 Antibody ® (monoclonal, 9E2)
Cat # M04201
Real IHC data
Anti-Integrin beta 5/ITGB5 Antibody ®
Cat # A04201-1
M04201 will render with an IHC image from a human lung cancer paraffin section (M04201 IHC caption). A04201-1 will render with an IHC image from a human rectal cancer paraffin section; its IF image uses A431 cells (A04201-1 IHC and IF captions).
Which to pick: For human tissue IHC, choose mouse monoclonal M04201: its image documents EDTA pH 8.0 retrieval and 2 μg/ml primary antibody in a paraffin section; the fixative is unreported (M04201 catalog entry and IHC caption). For IF/ICC, choose rabbit A04201-1, listed for both applications and illustrated in A431 cells at 4 μg/ml; its separate IHC image also uses a paraffin section with an unreported fixative (A04201-1 catalog entry, IF caption and IHC caption). For broader listed species reactivity, M04201-1 includes human, monkey and rat, though no IHC image is supplied for it; A04201-1 lists human and mouse, while its supplied IHC image documents human tissue only (catalog entries and A04201-1 IHC caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P18084 (ITB5_HUMAN, Integrin beta-5).
- Human Protein Atlas. ITGB5 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. ITGB5 subcellular location (ICC-IF): Localized to the plasma membrane and mitochondria..
- Human Protein Atlas. ITGB5 antibody validation summary (2 antibodies).
- A high-affinity anti-ITGB5 nanobody for hepatocellular carcinoma: Antitumor efficacy and tumor microenvironment reprogramming. The Journal of biological chemistry 2026 — PMC13570297.
- ROS-mediated ITGB5 promotes tongue squamous cell carcinoma metastasis through epithelial mesenchymal transition and cell adhesion signal pathway. Journal of cancer research and clinical oncology 2024 — PMC11344732.
- ITGB5 is a prognostic factor in colorectal cancer and promotes cancer progression and metastasis through the Wnt signaling pathway. Scientific reports 2025 — PMC11914080.
- Integrin-β5, a miR-185-targeted gene, promotes hepatocellular carcinoma tumorigenesis by regulating β-catenin stability. Journal of experimental & clinical cancer research : CR 2018 — PMC5793391.
- PubMed PMID:2328726 — UniProt-cited evidence.
- PubMed PMID:2371275 — UniProt-cited evidence.
- PubMed PMID:2211615 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ITGB5 (Integrin beta-5). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC); cell membrane location (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet M04201). Validated IHC antibody: M04201 at 2μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Breast (Glandular cells); negative Bone marrow. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet M04201); 2. Fixation: Image fixative and duration unreported (datasheet M04201); 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 M04201); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M04201); 8. Primary antibody: Mouse monoclonal (clone 9E2) anti-ITGB5, 2μg/ml (datasheet M04201); 9. Primary incubation: Overnight at 4 °C (datasheet M04201). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Lung macrophage staining can complicate cell attribution (HPA tissue IHC); Isoform / epitope: No isoforms annotated; extracellular and cytoplasmic regions differ (UniProt); Regulation: Expression regulation is not annotated (UniProt). Evidence: UniProt P18084, HPA tissue IHC · Enhanced, HPA ICC-IF · plasma membrane and mitochondria., Datasheet M04201, PMC13570297, PMC11344732. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ITGB5/immunohistochemistry-ihc-guide.