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SH3BP4 / SH3 domain-binding protein 4 · IHC design guide
Design Immunohistochemistry for SH3BP4
Plan chromogenic SH3BP4 IHC-P around mainly cytoplasmic tissue staining (HPA tissue IHC). The catalog antibody was used at 5 μg/mL in human lung tissue (datasheet); interpret staining cautiously because its concordance with RNA expression is low (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
SH3BP4 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 | Mainly cytoplasmic in tissue (HPA tissue IHC) | |
| Staining pattern | Medium staining in pancreatic exocrine cells; mainly cytoplasmic (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Bronchus+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Staining has low concordance with RNA expression (HPA tissue IHC) | |
| Regulation | Expression regulation is not established (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage is unknown (UniProt) |
Section 1
Recommended SH3BP4 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet) with a published SH3BP4 paraffin-section protocol (PMC6391711).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A09036); 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 Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear 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 anti-SH3BP4, 5 μg/mL (datasheet A09036) |
| 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 | SH3BP4-positive staining in respiratory epithelial cells of bronchus (HPA tissue IHC: Medium). HPA tissue profile: Mainly cytoplasmic expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 at 95–98 °C for 20 min (page antigen retrieval). The article does not specify which retrieval buffer was used for SH3BP4 (PMC6391711).
Section 2
What Is the Expected SH3BP4 Staining Pattern?
SH3BP4 is associated with transferrin receptor-containing clathrin-coated pits and vesicles, with possible nuclear localisation; it has no transmembrane segment (UniProt Q9P0V3). Expect mainly cytoplasmic staining in several tissues, including the epithelial and glandular cell populations listed by HPA (HPA: tissue IHC). Treat the pattern as provisional: HPA rates tissue staining Uncertain because antibody staining has low consistency with RNA expression (HPA: reliability).
| Cytoplasmic staining in pancreatic exocrine glandular cells or bronchial respiratory epithelial cells. | This fits HPA's Medium staining in those cell populations and its mainly cytoplasmic tissue profile (HPA: pancreas, bronchus, tissue IHC). Vesicle-associated staining is biologically plausible (UniProt Q9P0V3: localisation), but tissue IHC alone cannot identify the stained structures as clathrin-coated vesicles. |
| Strong staining confined to the plasma membrane, with little cytoplasmic signal. | Review this as a compartment mismatch: SH3BP4 is associated with coated pits and vesicles and lacks a transmembrane segment (UniProt Q9P0V3: localisation, topology). A membrane edge signal alone does not establish SH3BP4 specificity; compare it with the expected cytoplasmic pattern (HPA: tissue IHC). |
| Prominent staining in adipocytes or liver cholangiocytes. | These particular cell populations are reported as Not detected (HPA: adipose adipocytes; liver cholangiocytes). Their staining warrants checks for off-target binding or endogenous chromogenic activity (general IHC practice). HPA's Uncertain reliability prevents treating either population as a definitive negative control (HPA: reliability). |
| Diffuse colour across cells and surrounding tissue, without a recognisable cell pattern. | This is difficult to score as SH3BP4: HPA describes mainly cytoplasmic cellular staining (HPA: tissue IHC). Review the no-primary control, blocking, washes and detection conditions for nonspecific background (general IHC practice) before comparing tissue intensity. |
| No signal in pancreatic exocrine glandular cells. | HPA reports Medium staining in this population, so an absent result calls for a run-level check (HPA: pancreas). Confirm tissue morphology, antigen retrieval, primary antibody conditions and chromogen development against the assay controls (general IHC practice); HPA's Uncertain rating limits any conclusion from one negative slide (HPA: reliability). |
💡Expected SH3BP4 appearanceA plausible positive is Medium, mainly cytoplasmic staining in pancreatic exocrine glandular or bronchial respiratory epithelial cells (HPA: pancreas, bronchus, tissue IHC); prominent adipocyte staining would be discordant with HPA's Not detected observation (HPA: adipose adipocytes), subject to its Uncertain reliability (HPA: reliability).
| Compartment and topology | SH3BP4 associates with clathrin-coated pits and cytoplasmic vesicles, has no transmembrane segment, and may also localise to the nucleus (UniProt Q9P0V3). Read cytoplasmic staining first against the HPA tissue pattern; do not automatically reject a nuclear component, but do not call nuclear-only staining confirmed from these tissue data (HPA: tissue IHC). |
| Choice of reference cells | Pancreatic exocrine glandular and bronchial respiratory epithelial cells are Medium; adipocytes and liver cholangiocytes are Not detected in the supplied tissue observations (HPA: pancreas, bronchus, adipose, liver). Compare the named cell populations, not whole-organ colour, and retain HPA's Uncertain reliability when judging discordance (HPA: reliability). |
| Antibody evidence | Both listed antibodies, HPA037533 and HPA037534, have Uncertain IHC status (HPA: antibodies). Concordant morphology and assay controls strengthen interpretation in a particular run (general IHC practice), but these entries do not establish an independently verified SH3BP4 staining pattern. |
| Isoforms and epitope information | UniProt lists 2 SH3BP4 isoforms and SH3, ZU5 and SH3 domains (UniProt Q9P0V3: isoforms, domains). The supplied record gives no antibody epitope position, so it cannot predict which isoform a given IHC signal represents or prescribe an epitope-specific antigen retrieval condition. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected epithelial or glandular cells show no chromogenic signal. | The run may have inadequate retrieval, primary antibody binding or detection (general IHC practice); absence alone does not resolve specificity while HPA reliability is Uncertain (HPA: reliability). | Check a same-run positive reference cell population, retrieval record, reagent order and detection controls; HPA reports Medium pancreatic exocrine glandular staining (HPA: pancreas; general IHC practice). |
| Signal is uniformly diffuse or obscures cell borders. | Nonspecific binding or excess detection signal can obscure localisation (general IHC practice). This appearance is hard to reconcile with HPA's mainly cytoplasmic cellular profile (HPA: tissue IHC). | Inspect the no-primary control, blocking and wash steps, then adjust assay conditions using the antibody's actual IHC-P instructions (general IHC practice); no target-specific dilution is supplied here. |
| Staining appears predominantly at cell surfaces. | A sharp surface outline needs scrutiny because SH3BP4 has no transmembrane segment and is associated with coated pits and vesicles (UniProt Q9P0V3: topology, localisation). | Compare cell borders with cytoplasmic staining on the same slide and with a no-primary control (general IHC practice); avoid scoring a surface-only outline as confirmed SH3BP4. |
| Nuclear staining dominates the section. | Nuclear localisation is possible, but HPA describes the tissue IHC profile as mainly cytoplasmic (UniProt Q9P0V3: localisation; HPA: tissue IHC). | Check whether cytoplasmic signal accompanies it, review the no-primary control and score nuclear staining separately (general IHC practice); do not dismiss or validate it solely from HPA tissue IHC. |
| Adipocytes or cholangiocytes stain strongly. | HPA reports these cell populations as Not detected; off-target binding or endogenous detection activity is possible (HPA: adipose adipocytes, liver cholangiocytes; general IHC practice). | Compare the no-primary control and detection-only appearance, then verify cell identity and assay controls (general IHC practice). Treat the mismatch as a warning because HPA tissue reliability is Uncertain (HPA: reliability). |
| Can this IHC-P pattern be used to interpret IF/ICC? | The supplied HPA subcellular entry has no main location or ICC-IF image-bearing cell lines (HPA: subcellular ICC-IF); tissue IHC is rated Uncertain (HPA: reliability). | Use the separate IF/ICC guide for that application. Treat UniProt's vesicle and possible nuclear locations as hypotheses for image review (UniProt Q9P0V3: localisation), without converting this IHC-P guide into an IF/ICC protocol. |
Experiment evidence
IHC & IF images showing SH3BP4 staining
A09036 · IHCImmunohistochemistry of SH3BP4 in human lung tissue with SH3BP4 antibody at 5 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000130147-SH3BP4/tissue/cerebral+cortex#imid_8987690
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000130147-SH3BP4/tissue/colon#imid_8984304
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000130147-SH3BP4/tissue/liver#imid_8984297
HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000130147-SH3BP4/tissue/pancreas#imid_8984248
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for SH3BP4 IHC & IF
🧪Run bronchus first and check that respiratory epithelial cells stain (HPA: bronchus respiratory epithelial cells, Medium). Run adipose tissue as the negative comparator (HPA: adipocytes, Not detected); on the bronchus slide, use cells with background-level signal as internal negatives only after confirming their identity, since the supplied bronchus row does not identify a negative cell type (HPA: bronchus respiratory epithelial cells, Medium).
Positive control tissue: Bronchus (Respiratory epithelial cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for SH3BP4; derive a cell-line control from the positive tissue's cell type (Respiratory epithelial cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary (secondary-only) control, an isotype control matched to the primary antibody’s host species and clonality, and a knockout or peptide-block control where available (standard IHC practice). Quench endogenous peroxidase for chromogenic detection and check the bronchus no-primary section for residual signal (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the A09036 human lung IHC caption does not state a fixative (selected-SKU caption: fixative unreported). Retrieval dependency is also unreported; optimize antigen retrieval for paraffin sections with the controls above (standard IHC practice). The supplied evidence does not establish that frozen sections or IF are easier; assess bronchus background carefully when scoring epithelial staining (HPA: bronchus respiratory epithelial cells, Medium).
HPA tissue IHC evidence for SH3BP4
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Low 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 |
|---|---|---|---|---|
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | Medium | Protein (IHC) | HPA → |
| Colon | Glandular cells | Medium | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Medium | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Endometrium | Cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced SH3BP4 IHC Tips
Troubleshoot SH3BP4 staining in paraffin sections by assessing retrieval, cell type, compartment, controls, and scoring together (UniProt Q9P0V3; HPA tissue IHC).
How should I adjust retrieval when SH3BP4 staining is weak or uneven?
Start with Tris-EDTA at pH 9.0 for heat-induced retrieval at 95–98 °C for 20 min (page retrieval setting). If staining remains weak, compare a shorter heating period with the stated setting on adjacent sections, keeping antibody concentration and detection constant (standard IHC practice). Check that sections stay submerged, cool consistently, and retain intact tissue; patchy staining can reflect uneven heating or section loss (standard IHC practice). Assess respiratory epithelial cells in bronchus alongside a no-primary control, but treat staining as provisional because tissue IHC consistency is uncertain (HPA: medium in bronchial respiratory epithelial cells; HPA: reliability uncertain).
Can fixation explain faint or variable SH3BP4 staining in paraffin sections?
SH3BP4-specific sensitivity to fixation is unknown, so do not assign a faint result to a particular fixative or fixation duration (supplied evidence: no target-specific fixation data). The selected A09036 lung IHC caption reports 5 µg/mL antibody but does not state a fixative (selected A09036 tissue-IHC caption). Record each specimen’s fixative and processing history, then compare similarly processed sections while holding retrieval, antibody incubation, and detection constant (standard IHC practice). Include a no-primary control and examine tissue preservation before interpreting differences between specimens as changes in SH3BP4 expression (standard IHC practice).
Which SH3BP4 staining pattern should count as plausible in tissue?
Prioritize cytoplasmic staining when reviewing tissue sections, because that is the reported predominant IHC pattern across several tissues (HPA: mainly cytoplasmic expression). Fine peripheral or punctate cytoplasmic signal can be biologically plausible because SH3BP4 associates with transferrin receptor-containing clathrin-coated pits and vesicles (UniProt Q9P0V3: subcellular location). Nuclear signal is possible, but should be assessed separately and supported by reproducibility and controls rather than accepted from a single section (UniProt Q9P0V3: may localize to nucleus; standard IHC practice). Compare the pattern within identified cell types: bronchial respiratory epithelium has medium reported staining, whereas lung macrophages have low reported staining (HPA tissue IHC).
Could isoforms or epitope accessibility alter my SH3BP4 IHC result?
Check the antibody immunogen and epitope documentation before interpreting a negative section: SH3BP4 has 2 recorded isoforms, but the supplied evidence does not map this antibody’s epitope (UniProt Q9P0V3: isoforms; supplied antibody evidence). Its 963-residue chain contains SH3 domains at residues 55–114 and 654–724, plus a ZU5 domain at 317–454 (UniProt Q9P0V3: domains and processing). Several phosphoserines are recorded, including residues 131 and 246, without evidence here that they affect IHC binding (UniProt Q9P0V3: modified residues). If antibodies with documented, distinct epitopes are available, compare their spatial patterns on adjacent sections and interpret agreement alongside no-primary controls (standard IHC practice).
How should I adapt SH3BP4 localisation checks to multiplex IF?
For the separate IF/ICC workflow, pair SH3BP4 with a marker that identifies the expected cell type, such as a validated respiratory epithelial marker when examining bronchus (HPA: medium staining in bronchial respiratory epithelial cells; standard IF practice). Choose spectrally separated fluorophores and check unstained tissue first; a longer-wavelength SH3BP4 channel can help when shorter-wavelength tissue autofluorescence is prominent (standard IF practice). Use permeabilisation appropriate for an intracellular epitope, because SH3BP4 has no transmembrane segment and is associated with cytoplasmic vesicles and coated pits (UniProt Q9P0V3: topology and subcellular location). Include single-stain and no-primary controls before assigning puncta or apparent nuclear signal to SH3BP4 (standard IF practice; UniProt Q9P0V3: possible nuclear localisation).
How can I distinguish SH3BP4 signal from chromogenic background?
Run a no-primary control through the same peroxidase detection and chromogen steps to identify reagent-derived colour (standard IHC practice). Apply a peroxidase block before DAB development and compare the control with the stained section; these are general chromogenic workflow steps, not SH3BP4-specific validation (standard IHC practice). If diffuse colour obscures cellular detail, adjust blocking, antibody concentration, wash stringency, or development time one variable at a time (standard IHC practice). Judge any improvement in bronchial respiratory epithelium against morphology and controls, since reported medium staining has uncertain overall IHC reliability (HPA: bronchial respiratory epithelial cells medium; HPA: reliability uncertain).
What is a defensible way to quantify SH3BP4 IHC across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell type and compartment before scoring, then report the percentage of positive cells and staining intensity for each specimen (standard IHC practice; HPA: mainly cytoplasmic expression). An H-score combines intensity categories with their positive-cell percentages on a 0–300 scale; keep thresholds and imaging conditions consistent across the comparison (standard IHC practice). Normalize counts to viable cells of the same type, or express positive-cell density per mm² of viable annotated tissue when cell counts are impractical (standard IHC practice). Score bronchial respiratory epithelial cells separately from lung macrophages because their reported staining levels differ, and flag the uncertain reliability of the reference pattern (HPA tissue IHC).
When should an apparent SH3BP4-positive area be considered artefactual?
Check whether the signal follows intact cell boundaries and the expected predominantly cytoplasmic pattern before calling an area positive (HPA: mainly cytoplasmic expression; standard IHC practice). Treat isolated staining at section edges, folds, or necrotic regions cautiously, especially if a no-primary control shows similar colour (standard IHC practice). Investigate diffuse peroxidase-derived DAB signal with the blocked control, and distinguish bronchial respiratory epithelial cells from macrophages before comparing staining levels (standard IHC practice; HPA: bronchial respiratory epithelial cells medium, lung macrophages low). Nuclear staining may be plausible, but requires reproducibility and supporting controls because the tissue IHC reference has uncertain reliability (UniProt Q9P0V3: possible nuclear localisation; HPA: reliability uncertain).
Boster reagents
Best SH3BP4 / SH3 domain-binding protein 4 IHC Antibodies
A09036 has real IHC data from human lung tissue (image caption). Its listed reactivity covers human, mouse, and rat; no IF data are supplied (catalog: reactivity; IF images).
Real IHC data
Anti-SH3 domain-binding protein 4 SH3BP4 Antibody
Cat # A09036
A09036 is listed for IHC-P, with an IHC image from human lung tissue at 5 μg/mL (catalog: applications; image caption). Human, mouse, and rat are listed as reactive species, but the supplied IHC image documents human tissue only (catalog: reactivity; image caption).
Which to pick: For tissue IHC, choose A09036: it lists IHC-P and its own image caption shows human lung tissue at 5 μg/mL; the fixative is unreported (catalog: applications; image caption). No listed SKU supports an IF/ICC selection because A09036 has no IF application or IF image (catalog: applications; IF images). For cross-species work, A09036 lists human, mouse, and rat reactivity, although its IHC image covers human only; clonality is unreported (catalog: reactivity and clone; image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9P0V3 (SH3B4_HUMAN, SH3 domain-binding protein 4).
- Human Protein Atlas. SH3BP4 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. SH3BP4 subcellular location (ICC-IF): Highest expression in GAMG: 105.6 nTPM.
- Human Protein Atlas. SH3BP4 antibody validation summary (2 antibodies).
- SH3BP4 Regulates Intestinal Stem Cells and Tumorigenesis by Modulating β-Catenin Nuclear Localization. Cell reports 2019 — PMC6391711.
- PubMed PMID:10644451 — UniProt-cited evidence.
- PubMed PMID:16325581 — UniProt-cited evidence.
- PubMed PMID:15815621 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for SH3BP4 (SH3 domain-binding protein 4). Expected localisation Mainly cytoplasmic in tissue (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A09036 at 5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Bronchus (Respiratory epithelial cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet A09036); 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 Tris-EDTA buffer, pH 9.0 , 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 anti-SH3BP4, 5 μg/mL (datasheet A09036); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Staining has low concordance with RNA expression (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage is unknown (UniProt); Regulation: Expression regulation is not established (UniProt). Evidence: UniProt Q9P0V3, HPA tissue IHC · Uncertain, HPA cell-line RNA · Highest expression in GAMG: 105.6 nTPM, Datasheet A09036, PMC6391711. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/SH3BP4/immunohistochemistry-ihc-guide.