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GRB14 / Growth factor receptor-bound protein 14 · IHC design guide
Design Immunohistochemistry for GRB14
Plan GRB14 staining in paraffin sections using the reported granular cytoplasmic and membranous tissue pattern (HPA tissue IHC). The guide covers controls, detection and scoring while accounting for the uncertain agreement between antibody staining and RNA expression (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
GRB14 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 | Granular cytoplasm and membrane in tissues (HPA tissue IHC) | |
| Staining pattern | Granular cytoplasmic and membranous staining in glandular cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A04177-2) | |
| Positive control | Caudate+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 paraffin section fixation consistent (standard IHC practice; not target-specific) | |
| Caveat | Staining has low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | Insulin shifts GRB14 to the plasma membrane (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended GRB14 IHC & IF Protocols
Compare the catalog antibody’s EDTA pH 8.0 IHC protocol (datasheet: A04177-2) with published GRB14 staining of mouse embryos, gastric tissue and rodent retina (PMC11441139; PMC12627322; PMC2763493).
| Sample | Paraffin-embedded human renal cancer tissue; fixative not specified (datasheet A04177-2) |
| Fixation | Image fixative and duration unreported (datasheet A04177-2); 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 A04177-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A04177-2) |
| Primary antibody | Rabbit anti-GRB14, 2-5μg/ml (datasheet A04177-2) |
| Primary incubation | Overnight at 4 °C (datasheet A04177-2) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A04177-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | GRB14-positive staining in neuronal cells of caudate (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression with a granular pattern and membranous expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 for the catalog antibody (datasheet: A04177-2); evaluate citrate retrieval separately for the published gastric and retinal protocols (PMC12627322; PMC2763493).
Section 2
What Is the Expected GRB14 Staining Pattern?
GRB14 is a cytoplasmic protein also associated with endosome membranes; insulin stimulation can move it to the plasma membrane, and it has no transmembrane segment (UniProt Q14449). In tissue IHC, expect granular cytoplasmic and some membranous staining in selected cells, including epididymal glandular and neuronal cells (HPA: tissue IHC). Interpret cautiously: HPA rates the tissue pattern Uncertain because staining and RNA expression have low consistency (HPA: tissue IHC).
| Granular cytoplasmic staining, with some membrane staining, in epididymal glandular cells or neurons. | This fits the reported IHC pattern; epididymal glandular cells and neurons in the caudate or cerebral cortex have Medium staining (HPA: tissue IHC). Compare stained cells with tissue morphology and a counterstain. A plausible pattern supports identification, but the HPA tissue result remains Uncertain (HPA: tissue IHC). |
| Predominantly nuclear staining in a paraffin section, with little cytoplasmic signal. | That differs from the reported tissue IHC pattern and UniProt cytoplasmic location (HPA: tissue IHC; UniProt Q14449). Check morphology, detection controls and staining conditions before calling it GRB14. Nucleoplasm is an approved ICC-IF location, but that cell-based observation does not establish a nuclear tissue IHC pattern (HPA: subcellular ICC-IF). |
| Strong staining in adipocytes or lymph-node germinal-center cells. | Those cell populations were Not detected in the supplied tissue IHC data (HPA: tissue IHC). Unexpected signal raises a specificity or endogenous detection concern; review the cell identity and controls. Because HPA rates tissue IHC Uncertain, the discrepancy warrants investigation rather than an automatic false-positive label (HPA: tissue IHC). |
| Uniform color across cells, extracellular spaces or the whole section. | A field-wide haze is less persuasive than the reported cellular, granular cytoplasmic and membranous pattern (HPA: tissue IHC). General IHC practice: compare a no-primary control and examine blocking, washes and chromogen development. These checks identify possible detection background; they do not establish a GRB14-specific cause. |
| No staining in epididymal glandular cells despite an intact section. | HPA reports Medium staining in these cells, so absence merits a technical and tissue-identity check (HPA: tissue IHC). Confirm the expected cells are present, inspect the run control and review the IHC-validated antibody instructions. A negative section alone is not proof of absent GRB14, given the Uncertain tissue reliability (HPA: tissue IHC). |
💡Expected GRB14 appearanceA plausible positive is Medium, granular cytoplasmic staining with possible membrane signal in epididymal glandular cells or neurons; widespread acellular color or strong signal in HPA Not detected cell populations suggests a false-positive pattern requiring controls (HPA: tissue IHC, Uncertain).
| Localization and stimulation (UniProt Q14449) | GRB14 is cytoplasmic and associated with endosome membranes; insulin stimulation can shift it to the plasma membrane (UniProt Q14449). A membrane component can therefore be biologically plausible, but stimulation status is not provided for the HPA tissue sections. Do not use membrane staining alone to infer insulin activation. |
| Tissue and cell selection (HPA: tissue IHC) | HPA reports Medium staining in epididymal glandular cells and several neuronal populations, while adipocytes and lymph-node germinal-center cells were Not detected (HPA: tissue IHC). Select and score the named cell populations, rather than treating a whole organ as uniformly positive or negative. |
| Evidence strength and antibody agreement (HPA: tissue IHC; HPA: antibodies) | The overall tissue IHC reliability is Uncertain because antibody staining and RNA expression show low consistency (HPA: tissue IHC). Both supplied antibodies with IHC assessments, HPA035053 and CAB022294, are individually rated Uncertain (HPA: antibodies). Treat intensity and unexpected compartments as provisional until controls and an independent approach support them. |
| Does ICC-IF predict the paraffin IHC pattern? (HPA: subcellular ICC-IF) | ICC-IF reports approved nucleoplasm and cytosol locations and images from A-549, HEK293 and U2OS (HPA: subcellular ICC-IF). Tissue IHC instead reports granular cytoplasmic and membranous expression (HPA: tissue IHC). Read the two applications in their own context; the ICC-IF result is not a paraffin-section protocol or a reason to require nuclear IHC staining. |
| Protein forms and epitope knowledge (UniProt Q14449) | UniProt lists two GRB14 isoforms, no transmembrane segment and no signal peptide (UniProt Q14449). The supplied evidence does not map the IHC antibody epitope or its recognition of each isoform. Accordingly, do not assign a staining difference to isoform selectivity, shedding or a specific retrieval condition. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected epididymal cells show no signal (HPA: Medium in glandular cells). | The target cells may be absent from the examined area, or the IHC run may have failed; the result alone does not distinguish these explanations. | Confirm glandular-cell morphology and inspect a run control. Review the catalog antibody's IHC-P instructions for retrieval, dilution and detection; change one general workflow variable at a time. |
| Most of the section has diffuse color. | Nonspecific primary or detection binding, endogenous detection activity, or excessive chromogen development are general IHC possibilities. | Compare a no-primary control, check the detection system's endogenous-activity controls, and review blocking, washes and development. Seek a cellular pattern matching HPA's granular cytoplasmic or membranous description (HPA: tissue IHC). |
| Nuclear staining dominates the tissue section. | This differs from the supplied tissue IHC profile, although nucleoplasm is an approved ICC-IF location (HPA: tissue IHC; HPA: subcellular ICC-IF). | Check the counterstain, cell boundaries and no-primary control. Reassess with an independent specificity control before assigning nuclear GRB14 in tissue; keep the ICC-IF observation separate. |
| Adipocytes or germinal-center cells stain strongly. | HPA reports these cell populations as Not detected, so cell identification, cross-reactivity or detection background may explain the discrepancy (HPA: tissue IHC). | Verify the cell type and compare controls and another region of the section. Report the mismatch with HPA's Uncertain tissue reliability instead of converting a single stain into a new expression claim (HPA: tissue IHC). |
| Only a sharp membrane rim stains. | Plasma-membrane translocation is possible after insulin stimulation, but the stimulus status of the section is unknown (UniProt Q14449). | Check whether granular cytoplasmic signal or a credible cell-specific pattern accompanies the rim (HPA: tissue IHC). Review edge and background artifacts, then describe the observed compartment without claiming insulin activation. |
| Ovarian follicle cells are negative despite a high-expression tissue description. | UniProt describes high expression in ovary, whereas HPA reports follicle cells as Not detected by tissue IHC; the statements concern different levels of observation (UniProt Q14449; HPA: tissue IHC). | Record the sampled cell population and the IHC result separately from organ-level expression. Check controls before interpreting the discrepancy, and avoid using ovarian follicle cells as a guaranteed positive control. |
Experiment evidence
IHC & IF images showing GRB14 staining
A04177-2 · IHCIHC analysis of GRB14 using anti-GRB14 antibody (A04177-2). GRB14 was detected in paraffin-embedded section of human renal cancer tissue. He
A04177-2 · IFIF analysis of GRB14 using anti-GRB14 antibody (A04177-2). GRB14 was detected in immunocytochemical section of A549 cells. Enzyme antigen re
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000115290-GRB14/tissue/cerebral+cortex#imid_5867304
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000115290-GRB14/tissue/colon#imid_5867238
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000115290-GRB14/tissue/liver#imid_5867239
HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000115290-GRB14/tissue/pancreas#imid_5867198
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA035052; green = GRB14, blue = nucleus, red = microtubules.
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA035052; green = GRB14, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for GRB14 IHC & IF
🧪Run caudate first and expect staining in neuronal cells (HPA: Caudate, neuronal cells, Medium). Use adipose tissue as the negative tissue, where adipocytes are listed as not detected (HPA: Adipose tissue, adipocytes, Not detected). On the caudate slide, treat cells without specific staining as an internal background reference; the supplied HPA row does not establish which other cell types must be negative (HPA: Caudate, neuronal cells, Medium).
Positive control tissue: Caudate (Neuronal cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show GRB14 in A-549, HEK293, U2OS, with annotated localisation: Nucleoplasm (approved), Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a concentration-matched rabbit IgG isotype control alongside a GRB14 knockout biological negative (caption: rabbit anti-GRB14 primary; standard IHC practice). Quench endogenous peroxidase for DAB detection and inspect caudate neurons for pigment that could resemble staining or autofluoresce in IF (caption: DAB detection; standard IHC/IF practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected A04177-2 tissue-IHC caption does not state a fixative (caption: fixative not stated). Heat-mediated EDTA retrieval at pH 8.0 is a documented starting condition for paraffin sections, but its necessity has not been established by the supplied evidence (caption: heat-mediated EDTA retrieval). There is no matched frozen-section evidence to show that frozen sections are easier; HPA reports ICC-IF images but does not establish that IF is easier than IHC, and neuronal pigment in caudate can complicate signal assessment (HPA: A-549, HEK293 and U2OS ICC-IF images; HPA: Caudate neuronal cells, Medium; standard IHC/IF practice).
HPA tissue IHC evidence for GRB14
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Low 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 |
|---|---|---|---|---|
| Caudate | Neuronal cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | Medium | Protein (IHC) | HPA → |
| Cervix | Squamous epithelial cells | Medium | Protein (IHC) | HPA → |
| Duodenum | 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 → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced GRB14 IHC Tips
Troubleshoot GRB14 staining in paraffin sections by checking retrieval, compartment pattern, controls and scoring against the supplied IHC evidence.
Which antigen retrieval should I start with for GRB14 in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A04177-2). The selected tissue image used this retrieval before incubation with 2 µg/ml catalog antibody overnight at 4°C (caption A04177-2). If staining is weak, compare a more or less intense heating cycle on adjacent sections while keeping antibody concentration, detection and development conditions matched (standard IHC practice). Include a no-primary control to distinguish poor antigen exposure from background produced by the biotin-based detection workflow used in the image (caption A04177-2; standard IHC practice).
Could fixation explain weak or patchy GRB14 staining?
The selected image identifies a paraffin-embedded human renal cancer section but does not report its fixative, so target-specific fixation sensitivity is unknown (caption A04177-2). Record fixative, fixation duration and tissue processing for each specimen before comparing staining across batches (standard IHC practice). On adjacent sections, hold EDTA retrieval at pH 8.0 and antibody incubation at 2 µg/ml overnight at 4°C constant while assessing whether the patchiness tracks processing boundaries (datasheet A04177-2; standard IHC practice). Do not infer a GRB14-specific fixation effect from its phosphorylation sites, lack of a transmembrane segment or HPA tissue pattern (UniProt Q14449; HPA tissue IHC).
How should I assess cytoplasmic, membranous and nuclear GRB14 staining?
Expect cytoplasmic staining, potentially granular or membranous, when assessing tissue sections (UniProt Q14449 subcellular; HPA tissue IHC profile). GRB14 has no transmembrane segment, and insulin stimulation can move it toward the plasma membrane, so score a membrane rim separately from diffuse cytoplasmic signal (UniProt Q14449 topology and subcellular). HPA also reports approved nucleoplasm and cytosol locations from subcellular imaging; verify nuclear staining in paraffin sections with appropriate controls before treating it as established tissue localisation (HPA subcellular; standard IHC practice). Compare intact cells away from cut edges, and use the same compartment definitions across all slides and observers (standard IHC practice).
Could isoforms or epitope position change the GRB14 IHC pattern?
GRB14 has 2 listed isoforms and defined Ras-associating, PH and SH2 domains spanning residues 106–192, 234–342 and 439–535, respectively (UniProt Q14449). The supplied antibody caption does not identify its epitope, so neither isoform coverage nor sensitivity to local modifications can be assigned from that image (caption A04177-2). GRB14 includes reported phosphorylation at Ser372 and Ser375, which makes epitope mapping relevant if staining changes with experimental conditions (UniProt Q14449 modified residues; standard IHC practice). Check documented immunogen or epitope information before interpreting a negative section as absence of every GRB14 isoform (UniProt Q14449 isoforms; standard IHC practice).
How can IF help resolve ambiguous GRB14 localisation in tissue?
For tissue IF, multiplex GRB14 with a marker for the cell population being evaluated, then inspect both the merged image and each unmixed channel (standard IF practice). Select a fluorophore with minimal overlap with the specimen’s autofluorescence, and include single-label and no-primary controls to identify bleed-through or intrinsic signal (standard IF practice). Permeabilise sufficiently to expose cytosolic or endosome-facing epitopes; GRB14 has no transmembrane segment, but the supplied evidence does not locate this antibody’s epitope (UniProt Q14449 topology and subcellular; caption A04177-2). HPA reports nucleoplasm and cytosol localisation in ICC/IF, which can guide compartment review without establishing the appearance of a paraffin-section IF assay (HPA subcellular).
What should I check when GRB14 DAB staining is widespread?
The selected image used a biotinylated secondary, streptavidin-biotin detection and DAB, so assess each detection component when diffuse brown signal appears (caption A04177-2; standard IHC practice). Run a no-primary control and apply appropriate endogenous peroxidase and biotin controls or blocking for the chosen detection system (standard IHC practice). The reported section was blocked with 10% goat serum; compare blocking, washing and DAB development consistently across matched sections before changing antibody concentration (caption A04177-2; standard IHC practice). Exclude necrotic regions, damaged edges and pigment from GRB14 scoring, and require interpretable cellular morphology around any proposed granular or membranous pattern (HPA tissue IHC profile; standard IHC practice).
How should I quantify GRB14 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring because GRB14 can show cytoplasmic granular and membranous tissue staining (HPA tissue IHC profile). For chromogenic sections, report percentage of positive cells and staining intensity, or calculate an H-score from intensity categories on a 0–300 scale (standard IHC practice). Normalise positive-cell counts to all eligible cells in the specified population, or normalise positive-cell density to analysed tissue area in mm²; exclude necrosis and folds using the same rules for every slide (standard IHC practice). Keep retrieval, imaging, DAB development and scoring thresholds matched, and report compartments separately rather than combining a membrane rim with cytoplasmic staining (datasheet A04177-2; standard IHC practice).
When does GRB14 staining support a true positive call?
A credible positive shows reproducible cellular staining in an interpretable compartment, with low signal in the no-primary control and intact morphology (standard IHC practice). Cytoplasmic granular or membranous signal is compatible with the tissue profile, while nuclear staining deserves separate validation because its approved evidence comes from subcellular imaging (HPA tissue IHC profile; HPA subcellular). Check that staining belongs to the scored cell population: HPA lists medium signal in epididymal glandular cells but no detection in adipocytes, and rates its overall tissue IHC reliability as uncertain because staining and RNA agree poorly (HPA tissue IHC). Discount cut-edge staining, necrosis and endogenous detection signal before assigning biological meaning to an apparent GRB14 increase (standard IHC practice).
Boster reagents
Best GRB14 / Growth factor receptor-bound protein 14 IHC Antibodies
A04177-2 has IHC data from human renal cancer paraffin sections and IF data from A549 cells (catalog image captions); its listed reactivity is human (catalog reactivity).
Real IHC data
Anti-GRB14 Antibody ®
Cat # A04177-2
A04177-2 is listed for IHC and IF/ICC (catalog applications). Its IHC image shows human renal cancer paraffin sections, and its IF image shows A549 cells (catalog image captions).
Which to pick: For tissue IHC, choose A04177-2: its image shows paraffin sections with EDTA retrieval at pH 8.0; the fixative is unreported (catalog IHC image caption). For IF/ICC, the same SKU has an A549 cell IF image and an IF/ICC application listing (catalog IF image caption; catalog applications). No cross-species option is supported by this catalog: A04177-2 lists human reactivity only, and clonality is unreported (catalog reactivity; catalog clone field).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q14449 (GRB14_HUMAN, Growth factor receptor-bound protein 14).
- Human Protein Atlas. GRB14 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. GRB14 subcellular location (ICC-IF): Localized to the nucleoplasm and cytosol..
- Human Protein Atlas. GRB14 antibody validation summary (3 antibodies).
- The insulin resistance Grb14 adaptor protein promotes thyroid cancer ret signaling and progression. Oncogene 2012 — PMC3954818.
- Grb7, Grb10 and Grb14, encoding the growth factor receptor-bound 7 family of signalling adaptor proteins have overlapping functions in the regulation of fetal growth and post-natal glucose metabolism. BMC biology 2024 — PMC11441139.
- High expression of GRB14 is associated with cancer progression and poor prognosis in gastric cancer. Discover oncology 2025 — PMC12627322.
- Growth factor receptor-bound protein 14 undergoes light-dependent intracellular translocation in rod photoreceptors: functional role in retinal insulin receptor activation. Biochemistry 2009 — PMC2763493.
- PubMed PMID:8647858 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15815621 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for GRB14 (Growth factor receptor-bound protein 14). Expected localisation Granular cytoplasm and membrane in tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A04177-2). Validated IHC antibody: A04177-2 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Caudate (Neuronal cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human renal cancer tissue; fixative not specified (datasheet A04177-2); 2. Fixation: Image fixative and duration unreported (datasheet A04177-2); 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 A04177-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A04177-2); 8. Primary antibody: Rabbit anti-GRB14, 2-5μg/ml (datasheet A04177-2); 9. Primary incubation: Overnight at 4 °C (datasheet A04177-2). Watch for: Fixation: Keep paraffin section fixation consistent (standard IHC practice; not target-specific); Caveat: Staining has low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage is unspecified (UniProt); Regulation: Insulin shifts GRB14 to the plasma membrane (UniProt). Evidence: UniProt Q14449, HPA tissue IHC · Uncertain, HPA ICC-IF · nucleoplasm and cytosol., Datasheet A04177-2, PMC11441139, PMC12627322. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/GRB14/immunohistochemistry-ihc-guide.