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SRGAP3 / SLIT-ROBO Rho GTPase-activating protein 3 · IHC design guide
Design Immunohistochemistry for SRGAP3
Plan chromogenic SRGAP3 IHC in paraffin sections using the catalog antibody’s documented protocol (datasheet A06020-1). Compare staining in brain neuronal cells with adipocytes reported as undetected, and score signal by cell type and compartment (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
SRGAP3 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 in select tissues; cerebellar cells also show membrane staining (HPA tissue IHC) | |
| Staining pattern | Neurons and selected epithelial cells show cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A06020-1) | |
| Positive control | Cerebellum+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); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A06020-1) | |
| Caveat | Kidney isoform 3 is absent from brain; check epitope coverage (UniProt) | |
| Regulation | Brain-enriched; low in kidney (UniProt) | |
| Isoform / epitope | 3 isoforms; no signal peptide or propeptide; verify epitope coverage (UniProt) |
Section 1
Recommended SRGAP3 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet A06020-1) with a published SRGAP3 protocol for paraffin sections of low-grade glioma tissue (PMC9668894).
| Sample | Paraffin-embedded mouse brain tissue; fixative not specified (datasheet A06020-1) |
| Fixation | Image fixative and duration unreported (datasheet A06020-1); 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 A06020-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A06020-1) |
| Primary antibody | Rabbit anti-SRGAP3, 2-5 μg/ml (datasheet A06020-1) |
| Primary incubation | Overnight at 4 °C (datasheet A06020-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A06020-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | SRGAP3-positive staining in molecular layer cells - cytoplasm/membrane of cerebellum (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in a subset of tissues including urinary bladder, fallopian tube and cells of the CNS. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet A06020-1); citrate pH 6.0 microwave retrieval is a published alternative (PMC9668894).
Section 2
What Is the Expected SRGAP3 Staining Pattern?
In paraffin-section IHC, expect SRGAP3 mainly in the cytoplasm of selected cells, including CNS neurons and urothelial cells (HPA tissue IHC). Cerebellar molecular layer cells may show cytoplasmic or membrane-associated staining (HPA tissue IHC). The tissue profile has Enhanced reliability, with medium consistency between staining and RNA data (HPA tissue IHC). SRGAP3 has no transmembrane segment; UniProt does not assign a subcellular location (UniProt O43295 topology and subcellular annotation).
| Moderate staining appears in cerebral cortex neuronal cells, or in cerebellar molecular layer cell cytoplasm and membrane-associated regions (HPA tissue IHC). | These match reported positive patterns (HPA tissue IHC). Judge staining in the named cell population and compartment; a uniformly positive section is not required because HPA describes expression in a subset of tissues (HPA tissue IHC). |
| Strong nuclear staining dominates while cytoplasmic staining is absent. | Treat this as an unexpected pattern requiring verification: HPA describes tissue staining as cytoplasmic, and its ICC-IF main location is cytosol (HPA tissue IHC; HPA subcellular). Check morphology and compare an independently validated antibody if available (general IHC practice). |
| Adipocytes or adrenal glandular cells stain as strongly as the intended positive cells. | HPA reports those cell types as not detected (HPA tissue IHC). Consider antibody cross-reactivity or endogenous chromogen-generating activity; include a no-primary control to investigate the latter (general chromogenic IHC practice). |
| Broad, hazy chromogen covers cells and surrounding tissue without a clear cell pattern. | This does not resemble HPA's selected cytoplasmic staining (HPA tissue IHC). Background can arise from insufficient blocking, excessive antibody or detection reagent, or inadequate washing (general IHC practice). |
| No convincing signal appears in cerebral cortex neuronal cells or cerebellar molecular layer cells. | Both are reported at Medium staining level (HPA tissue IHC). First verify that the expected cells are present, then inspect the staining run and antibody conditions; one negative section alone cannot establish absent SRGAP3 expression (general IHC practice). |
💡Expected SRGAP3 appearanceCall a section positive when selected cells show moderate cytoplasmic staining, as reported for CNS cells and urothelium; diffuse staining across unrelated cells or dominant nuclear signal warrants checking (HPA tissue IHC; HPA subcellular).
| Tissue and cell selection | Cerebral cortex neurons, cerebellar molecular layer cells, proximal tubule cell bodies and urothelial cells are Medium-level examples; adipocytes and adrenal glandular cells are reported as not detected (HPA tissue IHC). Compare matched cell types when judging a result. |
| Strength of IHC evidence | The tissue profile is Enhanced, although antibody staining and RNA show medium consistency (HPA tissue IHC). Two rabbit polyclonals, HPA036959 and HPA044074, have Enhanced IHC status (HPA antibodies); interpret an isolated unexpected pattern cautiously. |
| Isoform and tissue context | UniProt reports high adult and fetal brain expression and low kidney expression; isoform 3 is reported in kidney but absent from brain (UniProt O43295 tissue specificity). The supplied evidence gives no isoform-specific IHC pattern, so kidney staining cannot identify an isoform. |
| Topology and processing | SRGAP3 has no transmembrane segment or signal peptide, and its annotated chain spans residues 1–1099 (UniProt O43295 topology and processing). These facts do not establish an extracellular staining pattern or a target-specific antigen retrieval requirement. |
| IF/ICC Q&A: What location should be expected? | HPA lists cytosol as the approved main location, with centrosome and basal body as additional approved locations (HPA subcellular). Its summary cautions that these data involve antibodies targeting proteins from multiple genes; HPA078079 has Uncertain ICC status (HPA antibodies). |
| Situation | Likely cause | Next action |
|---|---|---|
| A known-positive CNS section has no signal. | The expected neuronal population may be absent from the section, or the staining run may have failed (HPA tissue IHC; general IHC practice). | Confirm tissue identity and cell morphology; check the run's positive control, antibody dilution and detection steps (general IHC practice). |
| Signal is weak in a reported Medium-level cell type. | A weak run can be mistaken for low expression; the listed level describes HPA observations, not a guaranteed intensity for every assay (HPA tissue IHC; general IHC practice). | Compare a positive control stained in the same run and optimise antibody dilution or retrieval using the antibody's IHC instructions (general IHC practice). |
| Brown deposit appears broadly, including where no primary antibody was applied. | Endogenous detection activity or nonspecific reagent background may contribute (general chromogenic IHC practice). | Review the no-primary control, endogenous activity blocking and wash steps before interpreting tissue staining (general chromogenic IHC practice). |
| Staining concentrates in nuclei. | A dominant nuclear pattern conflicts with the cytoplasmic tissue profile and cytosolic ICC-IF location (HPA tissue IHC; HPA subcellular). | Recheck focus, counterstain and antibody specificity; compare with an independently validated IHC antibody if available (general IHC practice; HPA antibodies). |
| Adipocytes or adrenal glandular cells appear positive. | Those cell types are reported as not detected, making the pattern unexpected (HPA tissue IHC). Cross-reactivity or background is possible (general IHC practice). | Compare cell morphology with a reported positive tissue and inspect no-primary and negative-tissue controls (HPA tissue IHC; general IHC practice). |
| Centrosomal dots appear in IF/ICC and are being used to score IHC. | HPA lists centrosome and basal body as additional ICC-IF locations, while its tissue IHC profile describes selected cytoplasmic staining (HPA subcellular; HPA tissue IHC). | Score paraffin-section IHC against its tissue and cell pattern; use the separate IF/ICC guide for fluorescence interpretation (HPA tissue IHC; HPA subcellular). |
Experiment evidence
IHC & IF images showing SRGAP3 staining
A06020-1 · IHCIHC analysis of SRGAP3 using anti-SRGAP3 antibody (A06020-1). SRGAP3 was detected in a paraffin-embedded section of mouse brain tissue. Heat
A06020-1 · IHCIHC analysis of SRGAP3 using anti-SRGAP3 antibody (A06020-1). SRGAP3 was detected in a paraffin-embedded section of rat brain tissue. Heat m
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000196220-SRGAP3/tissue/cerebral+cortex#imid_8982938
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000196220-SRGAP3/tissue/colon#imid_8982864
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000196220-SRGAP3/tissue/liver#imid_8982857
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000196220-SRGAP3/tissue/kidney#imid_8982866
HPA · ICC-IF AF22Human Protein Atlas immunofluorescence, AF22, antibody HPA078079; green = SRGAP3, blue = nucleus, red = microtubules.
HPA · ICC-IF AF22Human Protein Atlas immunofluorescence, AF22, antibody HPA078079; green = SRGAP3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for SRGAP3 IHC & IF
🧪Run cerebral cortex first; neuronal cells should stain at Medium level (HPA: cerebral cortex, neuronal cells). Use adipose tissue as the negative tissue, where adipocytes are Not detected (HPA: adipose tissue, adipocytes); on the cortex slide, cells without specific staining should show counterstain and background only, with no convincing cytoplasmic DAB signal.
Positive control tissue: Cerebellum (Molecular layer cells - cytoplasm/membrane, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show SRGAP3 in AF22, U2OS, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), with annotated localisation: Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, a concentration-matched rabbit IgG control matched to the primary antibody’s clonality, and SRGAP3-knockout tissue where available to assess specificity (catalog antibody: rabbit primary; standard IHC controls). Block endogenous peroxidase before HRP/DAB detection and inspect pigment in brain sections that could be mistaken for chromogen (selected-SKU caption: HRP/DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported in the supplied evidence; the selected-SKU paraffin-section caption does not state the fixative (selected-SKU caption: fixative unreported). The demonstrated paraffin-section procedure used heat retrieval in EDTA pH 8.0, but the evidence does not establish that retrieval is required under every processing condition (selected-SKU caption: EDTA pH 8.0). There is no matched comparison showing that frozen sections or IF are easier; for brain IF, assess tissue autofluorescence with an unstained section (HPA: neuronal staining in cerebral cortex; standard IF practice).
HPA tissue IHC evidence for SRGAP3
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 |
|---|---|---|---|---|
| Cerebellum | Molecular layer cells - cytoplasm/membrane | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | Medium | Protein (IHC) | HPA → |
| Fallopian tube | Ciliated cells (ciliary rootlets) | Medium | Protein (IHC) | HPA → |
| Kidney | Proximal tubules (cell body) | Medium | Protein (IHC) | HPA → |
| Nasopharynx | Basal cells | Medium | 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 → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Duodenum | Endocrine cells | Not detected | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced SRGAP3 IHC Tips
Use the documented paraffin-section conditions as a starting point, then assess SRGAP3 staining against cell type, compartment and matched controls.
What retrieval should I use when SRGAP3 staining is weak in paraffin sections?
Start with heat-mediated EDTA retrieval at pH 8.0 for paraffin sections (datasheet A06020-1). The documented mouse brain image used that retrieval, followed by 10% goat serum blocking and 2 μg/ml primary antibody overnight at 4°C (caption A06020-1). If signal is weak, compare retrieval heating times on adjacent sections while keeping antibody concentration and detection constant (standard IHC practice). Include a no-primary control and inspect tissue integrity, since excessive heating can damage morphology or raise nonspecific staining (standard IHC practice). Record the heating conditions used: the caption specifies the buffer and pH but gives no retrieval time (caption A06020-1).
Could fixation explain weak or uneven SRGAP3 IHC staining?
Target-specific fixation sensitivity is unknown, and the selected paraffin-section image does not report its fixative (caption A06020-1). Compare sections with documented, matched fixation and processing histories before attributing signal differences to SRGAP3 abundance (standard IHC practice). On adjacent sections, hold EDTA retrieval at pH 8.0 and the starting antibody concentration at 2 μg/ml constant (datasheet A06020-1; caption A06020-1). Examine morphology alongside staining because delayed or uneven fixation can produce patchy antigen preservation in tissue sections (standard IHC practice). Neither tissue staining patterns nor SRGAP3 domain and phosphorylation annotations establish a fixation effect for this antibody (HPA tissue IHC; UniProt O43295).
Which cellular staining patterns are credible for SRGAP3 in tissue sections?
Prioritise cytoplasmic staining in the relevant cells: the tissue profile reports cytoplasmic expression, including CNS cells (HPA tissue IHC). Cerebellar molecular-layer cells show medium cytoplasmic or membrane-associated staining, while cerebral cortical neuronal cells show medium staining (HPA tissue IHC). SRGAP3 has no annotated UniProt subcellular location or transmembrane segment, so a crisp cell-surface outline alone needs independent validation (UniProt O43295 topology). The subcellular atlas reports cytosol plus centrosome and basal body, but cautions that those assignments draw on antibodies targeting multiple genes (HPA subcellular). Compare candidate staining with cell morphology and matched controls rather than treating that atlas pattern as proof in chromogenic sections (standard IHC practice).
How could isoforms or epitope placement change the IHC result?
SRGAP3 has 3 annotated isoforms, but the selected caption supplies no antibody epitope, so isoform coverage cannot be assigned (UniProt O43295; caption A06020-1). Its F-BAR domain spans residues 19–314, Rho-GAP 506–694, and SH3 744–803 (UniProt O43295). Request the immunogen or mapped epitope before interpreting absent staining as loss of all isoforms, particularly when comparing brain and kidney (standard IHC practice; UniProt O43295 tissue specificity). Isoform 3 is reported in kidney but absent from brain, whereas overall expression is high in adult and fetal brain and low in kidney (UniProt O43295 tissue specificity). Compare sections with an independently validated antibody to a defined region if isoform discrimination is essential (standard IHC practice).
How should I check SRGAP3 localisation by multiplex IF?
Use a marker for the expected cell population alongside SRGAP3; neuronal cells in cerebral cortex and molecular-layer cells in cerebellum are reported staining populations (HPA tissue IHC). Choose spectrally separated fluorophores and favour a far-red SRGAP3 channel when tissue autofluorescence compromises shorter-wavelength channels (standard IF practice). Because the antibody epitope is unspecified, optimise mild permeabilisation only if access to an intracellular epitope is needed, then compare signal and morphology (caption A06020-1; standard IF practice). The reported cytosol, centrosome and basal-body locations provide comparison points but carry a multi-gene-antibody caution (HPA subcellular). Run single-colour and no-primary controls to distinguish bleed-through or background from colocalisation (standard IF practice).
What should I change if DAB background obscures SRGAP3 staining?
First inspect a no-primary section and the tissue margins to separate reagent background from cell-associated staining (standard IHC practice). The documented chromogenic workflow used 10% goat serum blocking, peroxidase-conjugated secondary antibody for 30 minutes at 37°C, and DAB development (caption A06020-1). Add an appropriate endogenous-peroxidase block and control DAB development time when diffuse brown signal persists; these are general chromogenic IHC steps (standard IHC practice). If background follows the primary antibody, titrate around the documented 2 μg/ml starting concentration while keeping retrieval at EDTA pH 8.0 (caption A06020-1; datasheet A06020-1). Compare staining with a morphologically intact region on the same section (standard IHC practice).
How should I quantify SRGAP3 staining across tissue samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, since SRGAP3 tissue staining varies by cell type (HPA tissue IHC). For chromogenic sections, record percentage of positive target cells and staining intensity, then calculate an H-score consistently across slides (standard IHC practice). If distribution matters more than intensity, report positive-cell density per mm² of viable analysed tissue (standard IHC practice). Normalise each measure to the number of eligible cells or viable tissue area, and use matched section thickness, exposure and DAB development conditions (standard IHC practice). Keep neuronal and non-neuronal regions separate when their cellular composition differs, and score blinded to sample group where feasible (standard IHC practice).
How can I distinguish true SRGAP3 signal from an artefact?
A credible pattern follows cells and compartments with independent support: cortical neurons and cerebellar molecular-layer cells are reported positive, with cytoplasmic or membrane-associated cerebellar staining (HPA tissue IHC). Diffuse nuclear signal or a uniform cell-surface rim needs scrutiny because UniProt gives no subcellular annotation and no transmembrane segment (UniProt O43295). Exclude section-edge staining and necrotic areas when judging distribution, and inspect a no-primary control for nonspecific detection (standard IHC practice). Brown deposits that persist without primary antibody can indicate endogenous peroxidase or other detection background in a DAB workflow (standard IHC practice). Interpret kidney results cautiously because overall expression is reported low there and isoform 3 has a distinct brain-versus-kidney pattern (UniProt O43295 tissue specificity).
Boster reagents
Best SRGAP3 / SLIT-ROBO Rho GTPase-activating protein 3 IHC Antibodies
The catalog antibody has real IHC images from paraffin sections of human, mouse, and rat brain (catalog A06020-1 image captions). No IF/ICC data are supplied (catalog applications and images).
Real IHC data
Anti-SRGAP3 Antibody ®
Cat # A06020-1
A06020-1 is the only SKU shown (catalog). Its IHC images show paraffin sections of human, mouse, and rat brain (catalog A06020-1 image captions).
Which to pick: Choose A06020-1 for paraffin-section tissue IHC: its rabbit antibody has IHC listed and images from all three species (catalog applications, host, and image captions). The fixative is unreported (catalog A06020-1 image captions). No SKU can be recommended as IF/ICC-validated because IF/ICC is absent from the application list and no IF image is supplied (catalog applications and images).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O43295 (SRGP3_HUMAN, SLIT-ROBO Rho GTPase-activating protein 3).
- Human Protein Atlas. SRGAP3 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. SRGAP3 subcellular location (ICC-IF): Mainly localized to the cytosol. In addition localized to the centrosome and basal body. Caution: Based on antibodies targeting proteins from multiple genes..
- Human Protein Atlas. SRGAP3 antibody validation summary (3 antibodies).
- Structuring and validating a prognostic model for low-grade gliomas based on the genes for plasma membrane tension. Frontiers in neurology 2022 — PMC9668894.
- PubMed PMID:12447388 — UniProt-cited evidence.
- PubMed PMID:12195014 — UniProt-cited evidence.
- PubMed PMID:9455477 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for SRGAP3 (SLIT-ROBO Rho GTPase-activating protein 3). Expected localisation Cytoplasmic in select tissues; cerebellar cells also show membrane staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A06020-1). Validated IHC antibody: A06020-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Cerebellum (Molecular layer cells - cytoplasm/membrane); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded mouse brain tissue; fixative not specified (datasheet A06020-1); 2. Fixation: Image fixative and duration unreported (datasheet A06020-1); 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 A06020-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A06020-1); 8. Primary antibody: Rabbit anti-SRGAP3, 2-5 μg/ml (datasheet A06020-1); 9. Primary incubation: Overnight at 4 °C (datasheet A06020-1). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A06020-1); Caveat: Kidney isoform 3 is absent from brain; check epitope coverage (UniProt); Isoform / epitope: 3 isoforms; no signal peptide or propeptide; verify epitope coverage (UniProt); Regulation: Brain-enriched; low in kidney (UniProt). Evidence: UniProt O43295, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the cytosol. In addition localized to the…, Datasheet A06020-1, PMC9668894. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/SRGAP3/immunohistochemistry-ihc-guide.