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ARHGAP35 / Rho GTPase-activating protein 35 · IHC design guide
Design Immunohistochemistry for ARHGAP35
Plan ARHGAP35 IHC using the cytoplasmic tissue pattern and high staining in breast glandular and cerebellar Purkinje cells as benchmarks (HPA tissue IHC). For paraffin sections, start the IHC-validated antibody at 1:100–1:300 (datasheet: A03592) and score staining by cell type.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ARHGAP35 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 staining in most tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic signal across most tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Breast+4 more · see all | |
| Negative control | Liver |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A03592) | |
| Caveat | Signaling may shift protein to membrane ruffles (UniProt) | |
| Regulation | Expression regulation unreported (UniProt) | |
| Isoform / epitope | No annotated isoforms; one intact chain (UniProt) |
Section 1
Recommended ARHGAP35 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet) is paired with a published human gastric cancer tissue microarray procedure (PMC9342288).
| Sample | Paraffin-embedded human brain tissue; fixative not specified (datasheet A03592) |
| Fixation | Image fixative and duration unreported (datasheet A03592); 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-ARHGAP35, 1:100-1:300 (datasheet A03592) |
| 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 | ARHGAP35-positive staining in glandular cells of breast (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 retrieval at 95–98 °C for 20 min (page antigen retrieval).
Section 2
What Is the Expected ARHGAP35 Staining Pattern?
In paraffin-section IHC, expect predominantly cytoplasmic ARHGAP35 staining in many tissues, with strong staining reported in breast glandular cells, cerebellar Purkinje cells and cortical neurons (HPA: tissue IHC). ARHGAP35 has no transmembrane segment; UniProt also lists nuclear, cytoskeletal, basal-body and context-dependent membrane localization (UniProt Q9NRY4: localization and topology). The HPA tissue profile is Approved, with medium staining–RNA consistency and external verification pending (HPA: reliability).
| Clear cytoplasmic staining in breast or endometrial glandular cells, Purkinje cells, or cortical neurons. | This matches reported high staining in those cell populations and the predominantly cytoplasmic tissue profile (HPA: tissue IHC). Score the identified cells and compartment, rather than judging a section only by its overall color (general IHC practice). |
| Staining is concentrated in extracellular spaces or as granular deposits over the section, without a clear cellular pattern. | That distribution does not match the reported cytoplasmic tissue pattern and warrants an artifact check (HPA: tissue IHC; general IHC practice). Nuclear staining alone needs more careful interpretation: UniProt lists the nucleus, although HPA describes tissue IHC as mainly cytoplasmic (UniProt Q9NRY4: localization; HPA: tissue IHC). |
| Strong chromogenic staining appears in liver cholangiocytes while the chosen positive cells show little staining. | HPA reports ARHGAP35 as not detected in cholangiocytes, so verify cell identity and investigate antibody cross-reactivity or endogenous detection activity (HPA: liver IHC; general IHC practice). This comparison raises a concern; it does not, by itself, identify which mechanism produced the color. |
| Diffuse color covers cells and tissue spaces, obscuring cell boundaries. | The slide cannot support reliable compartment or cell-type scoring when background masks those features (general IHC practice). Compare a control lacking primary antibody and review blocking, washing and detection steps to locate nonspecific color (general IHC practice). |
| No signal is seen in a selected HPA high-staining tissue, including its reported positive cell population. | First confirm that the expected cells are present and identifiable; then assess staining-run controls, antibody conditions, antigen retrieval and detection (HPA: tissue IHC; general IHC practice). One negative section cannot establish biological absence, especially with HPA external verification still pending (HPA: reliability). |
💡Expected ARHGAP35 appearanceA positive IHC result shows discernible, predominantly cytoplasmic staining in the expected cells, with high staining possible in HPA-listed high populations; diffuse extracellular color or strong staining in reported negative cholangiocytes calls for investigation (HPA: tissue IHC; general IHC practice).
| Which compartment should guide IHC scoring? | Use the predominantly cytoplasmic HPA tissue pattern as the primary comparison (HPA: tissue IHC). UniProt also lists nucleus, cytoskeleton and basal body, plus membrane-ruffle localization after specified signaling, so an unusual intracellular pattern merits validation rather than automatic rejection (UniProt Q9NRY4: localization). |
| What distinctive ciliary result is documented? | HPA reports high staining at cilia tips in fallopian-tube and nasopharyngeal ciliated cells (HPA: tissue IHC). Evaluate that feature only where identifiable ciliated cells are present; absence of visible tips elsewhere is not evidence of target loss (general IHC practice). |
| How should tissue choice affect the comparison? | HPA reports low tissue RNA specificity and cytoplasmic protein expression in most tissues, but lists both high-staining cell populations and low-staining populations (HPA: tissue IHC). A low-staining population is a weaker sensitivity check than one reported high; cholangiocytes provide a reported not-detected comparison (HPA: tissue IHC). |
| How strong is the IHC validation? | Three listed antibodies have Approved IHC status: HPA055184, HPA056470 and CAB037311 (HPA: antibody validation). The tissue profile has medium staining–RNA consistency and awaits external verification (HPA: reliability); Approved should therefore be read with that qualification. |
| IF/ICC Q: Should nuclear bodies be expected in IHC? | HPA ICC-IF mainly localizes ARHGAP35 to nuclear bodies, with additional microtubule and aggresome localization (HPA: subcellular ICC-IF). That result is a useful modality-specific comparison; it does not replace the predominantly cytoplasmic paraffin-tissue IHC expectation (HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| A reported high-staining population is blank. | The expected cells may be absent from the section, or the staining run may have failed (HPA: tissue IHC; general IHC practice). | Confirm histology and run controls, then review antigen retrieval, primary-antibody conditions and detection using the selected antibody's IHC-P instructions (general IHC practice). |
| Cytoplasmic signal is weak only in a low-staining population. | HPA lists some populations as low, including ovarian follicle cells and skeletal-muscle myocytes (HPA: tissue IHC). | Compare an appropriate HPA high-staining population in the same run before interpreting weak color as a technical failure (HPA: tissue IHC; general IHC practice). |
| Cholangiocytes stain strongly. | This conflicts with HPA's not-detected cholangiocyte result; cross-reactivity or endogenous detection activity is possible (HPA: liver IHC; general IHC practice). | Check morphology and a control lacking primary antibody; review endogenous-enzyme blocking if the chromogenic detection system requires it (general IHC practice). |
| Color is diffuse or pools beyond cell boundaries. | Nonspecific background or chromogen deposit can obscure the expected cytoplasmic pattern (HPA: tissue IHC; general IHC practice). | Inspect controls and reagent deposits, then review blocking, washing and detection conditions before assigning compartment scores (general IHC practice). |
| Nuclear staining dominates the tissue section. | UniProt permits nuclear localization, while HPA describes tissue IHC as predominantly cytoplasmic; the discordance needs context (UniProt Q9NRY4: localization; HPA: tissue IHC). | Compare expected positive cells and controls, and seek independent antibody or orthogonal evidence before calling the nuclear pattern ARHGAP35-specific (general IHC practice). |
| A cilia-tip signal is missing. | The field may lack identifiable ciliated cells, or cilia may be poorly represented in the section (general IHC practice). | Confirm ciliated-cell morphology in fallopian-tube or nasopharyngeal tissue before assessing the HPA high cilia-tip pattern (HPA: tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing ARHGAP35 staining
A03592 · IHCImmunohistochemistry analysis of paraffin-embedded human brain tissue, using GRF-1 Antibody. The picture on the right is blocked with the sy
P30433 · IHCImmunohistochemical analysis of paraffin-embedded Human brain. Antibody was diluted at 1:100(4° overnight). High-pressure and temperature Tr
P30433 · IHCImmunohistochemistry analysis of paraffin-embedded human breast carcinoma, using GRF-1 (Phospho-Tyr1105) Antibody. The picture on the right
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000160007-ARHGAP35/tissue/cerebral+cortex#imid_16873775
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000160007-ARHGAP35/tissue/colon#imid_16873701
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000160007-ARHGAP35/tissue/liver#imid_16873702
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000160007-ARHGAP35/tissue/kidney#imid_16873703
HPA · ICC-IF HeLaHuman Protein Atlas immunofluorescence, HeLa, antibody HPA056470; green = ARHGAP35, blue = nucleus, red = microtubules.
HPA · ICC-IF HeLaHuman Protein Atlas immunofluorescence, HeLa, antibody HPA056470; green = ARHGAP35, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ARHGAP35 IHC & IF
🧪Run breast first and expect staining in glandular cells (HPA: Breast glandular cells, High). Run liver as the negative tissue and expect cholangiocytes to lack specific staining (HPA: Liver cholangiocytes, Not detected); on the breast slide, cells without specific staining should show only background signal, although the supplied HPA rows do not identify a validated internal negative cell type.
Positive control tissue: Breast (Glandular cells, HPA High)
Negative control tissue: Liver (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ARHGAP35 in HeLa, MCF-7, U2OS, hTERT-RPE1 (serum starved), Sperm, NIH 3T3, with annotated localisation: Nuclear bodies (approved) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, a control IgG matched to the primary antibody’s host species and isotype (and subclass if monoclonal), and a peptide-blocked primary as a biological specificity check (A03592 caption: staining blocked with synthesized peptide). Quench endogenous peroxidase on breast sections if using HRP chromogenic detection; an IF run should instead include an autofluorescence check.
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence, and the selected paraffin-section caption does not state a fixative (A03592 caption: paraffin-embedded human brain tissue; fixative unreported). Antigen retrieval dependence is unreported, so compare retrieval conditions during IHC optimization; the supplied evidence does not establish whether frozen sections or IF/ICC would be easier. No breast-specific artefact is documented in the supplied evidence.
HPA tissue IHC evidence for ARHGAP35
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium 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 |
|---|---|---|---|---|
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Endometrium | Glandular cells | High | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ARHGAP35 IHC Tips
Troubleshoot ARHGAP35 staining in paraffin sections by checking retrieval, compartment, cell identity and controls before comparing signal across samples.
Which retrieval conditions should I try first for ARHGAP35 in paraffin sections?
Use Tris-EDTA at pH 9.0 for heat-induced retrieval at 95–98 °C for 20 min (page retrieval rule). Let sections cool in the retrieval buffer, then process a matched control section alongside the sample so heating and detection conditions can be compared (standard IHC practice). If staining is weak, adjust heating time incrementally before testing an alternative buffer, and assess tissue integrity as well as signal (standard IHC practice). The human brain image for SKU A03592 shows paraffin-section staining and peptide blocking, but its caption does not report retrieval conditions or a fixative (A03592 tissue-IHC caption).
How should I assess whether fixation is limiting ARHGAP35 staining?
Target-specific sensitivity of ARHGAP35 staining to fixative choice or fixation duration is unknown from the supplied evidence (A03592 tissue-IHC caption; HPA tissue IHC). The A03592 human brain caption identifies paraffin-embedded tissue but does not state its fixative, so it cannot establish a formalin fixation protocol (A03592 tissue-IHC caption). Record the fixative and fixation duration for each sample, then compare sections processed with the same retrieval, antibody incubation and chromogenic detection conditions (standard IHC practice). If signal varies with processing history, compare matched tissue regions and inspect morphology before attributing the difference to ARHGAP35 biology (standard IHC practice).
Where should credible ARHGAP35 chromogenic staining appear?
Expect predominantly cytoplasmic staining across many tissues in the tissue IHC reference, while allowing context-dependent distribution rather than requiring one compartment in every cell (HPA tissue IHC; UniProt Q9NRY4 subcellular). ARHGAP35 is also annotated in the nucleus, cytoskeleton, cilium basal body and cell membrane, without a transmembrane segment (UniProt Q9NRY4 subcellular; UniProt Q9NRY4 topology). Integrin and SDC4 signalling with PKC phosphorylation can redistribute it toward membrane ruffles with polymerized actin (UniProt Q9NRY4 subcellular). Score cytoplasmic, nuclear and peripheral signal separately, and compare each pattern with cell morphology and adjacent negative controls before calling redistribution (standard IHC practice).
How can epitope placement change interpretation of ARHGAP35 staining?
Check the catalog antibody’s stated immunogen or epitope before comparing stains, because the supplied record gives no antibody epitope coordinates (A03592 tissue-IHC caption; UniProt Q9NRY4 record). The provided UniProt record lists a 1–1499 chain, 0 annotated isoforms, FF repeats, two pseudoGTPase domains and a Rho-GAP domain at residues 1249–1436 (UniProt Q9NRY4 processing, isoforms and domains). It also lists multiple modified residues, including phosphorylation at positions 308, 589, 770, 773 and 970, without establishing effects on this antibody’s staining (UniProt Q9NRY4 modified residues; A03592 tissue-IHC caption). If two antibodies disagree, compare their documented epitopes and controls before assigning an isoform-specific or phosphorylation-dependent explanation (standard IHC practice).
What should I check when comparing IHC with ARHGAP35 multiplex IF?
Treat IF as a separate validation context: the supplied A03592 image documents paraffin-section IHC with peptide blocking, without an IF protocol (A03592 tissue-IHC caption). In a multiplex IF experiment, pair ARHGAP35 with a validated marker for the expected cell population, such as a neuronal marker when examining the HPA-listed cerebral cortex neuronal cells, and assess channel overlap at cellular resolution (HPA tissue IHC; standard IF practice). Choose fluorophores and filter sets after checking tissue autofluorescence and single-stain controls (standard IF practice). Because ARHGAP35 has no transmembrane segment and has cytoplasmic and nuclear annotations, tune permeabilisation to expose intracellular epitopes without assuming membrane-sided accessibility (UniProt Q9NRY4 topology and subcellular; standard IF practice).
How can I reduce diffuse or misleading brown staining?
Compare a no-primary control with the full stain to identify signal from detection reagents, endogenous enzyme activity or pigment before changing the antibody concentration (standard chromogenic IHC practice). Include a peroxidase block for a peroxidase-based DAB workflow, and use the same blocking, wash and development times across control and test sections (standard chromogenic IHC practice). The A03592 human brain image includes a synthesized-peptide blocking comparison, which supports an antibody competition check for that image but does not define performance in other tissues (A03592 tissue-IHC caption). If background persists, shorten chromogen development or titrate antibody on matched sections while retaining a positive reference region (standard IHC practice).
How should I score ARHGAP35 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because the tissue reference reports broadly cytoplasmic expression while UniProt also annotates nuclear and peripheral locations (HPA tissue IHC; UniProt Q9NRY4 subcellular). For a defined population, report an H-score from intensity categories and the percentage of positive cells, or positive-cell density per mm² for spatial comparisons (standard IHC practice). Normalize counts to the number of eligible cells or measured viable tissue area, and keep section thickness, retrieval and chromogen exposure consistent across samples (standard IHC practice). Record cytoplasmic and nuclear scores separately, then document thresholds using control sections before applying them to the study set (standard IHC practice).
How do I distinguish true ARHGAP35 signal from artefact?
A credible pattern should align with cell morphology and the broad cytoplasmic tissue profile, while compartment changes need independent support because UniProt also annotates nuclear and membrane-associated locations (HPA tissue IHC; UniProt Q9NRY4 subcellular). HPA reports high staining in cerebellar Purkinje cells and cerebral cortex neuronal cells, whereas liver cholangiocytes are listed as not detected; these are comparison contexts, not universal controls for every specimen (HPA tissue IHC). Treat isolated staining at section edges, necrotic areas or sites of endogenous enzyme activity cautiously and inspect a no-primary control (standard chromogenic IHC practice). Peptide blocking in the A03592 brain image supports competition in that image, but confirm cell identity and reproducibility before interpreting a new tissue pattern (A03592 tissue-IHC caption; standard IHC practice).
Boster reagents
Best ARHGAP35 / Rho GTPase-activating protein 35 IHC Antibodies
Two antibodies have human paraffin-section IHC images (catalog image captions); both list Human, Mouse, and Rat reactivity and IF applications (catalog application and reactivity lists).
Real IHC data
Anti-GRF-1 ARHGAP35 Antibody
Cat # A03592
Real IHC data
Anti-Phospho GRF-1 (Tyr1105) Antibody
Cat # P30433
A03592 has a human paraffin-embedded brain IHC image with a peptide-blocked control and lists IF (A03592 image caption; catalog application list). P30433 has human paraffin-embedded brain and breast carcinoma IHC images with peptide-blocked controls and lists IF and ICC (P30433 image captions; catalog application list).
Which to pick: For tissue IHC, choose A03592 for ARHGAP35 or P30433 when Tyr1105 phosphorylation is the target; each has its own human paraffin-section IHC image, and neither caption reports the fixative (catalog titles; respective IHC image captions). For IF/ICC, P30433 lists both applications and is described as polyclonal, while A03592 lists IF and has no reported clonality; neither has an IF image in the payload (catalog application lists; P30433 dilution record; A03592 catalog record; catalog IF image lists). Both list Human, Mouse, and Rat reactivity for cross-species work, while their supplied IHC images show human tissue (catalog reactivity lists; respective IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9NRY4 (RHG35_HUMAN, Rho GTPase-activating protein 35).
- Human Protein Atlas. ARHGAP35 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ARHGAP35 subcellular location (ICC-IF): Mainly localized to the nuclear bodies. In addition localized to the aggresome and microtubules..
- Human Protein Atlas. ARHGAP35 antibody validation summary (3 antibodies).
- The m(6)A demethylases FTO and ALKBH5 aggravate the malignant progression of nasopharyngeal carcinoma by coregulating ARHGAP35. Cell death discovery 2024 — PMC10806234.
- Glucocorticoid-mediated induction of caveolin-1 disrupts cytoskeletal organization, inhibits cell migration and re-epithelialization of non-healing wounds. Communications biology 2021 — PMC8213848.
- Rho GTPase-activating protein 35 suppresses gastric cancer metastasis by regulating cytoskeleton reorganization and epithelial-to-mesenchymal transition. Bioengineered 2022 — PMC9342288.
- Ubiquitin ligase TRIM65 promotes colorectal cancer metastasis by targeting ARHGAP35 for protein degradation. Oncogene 2019 — PMC6756236.
- PubMed PMID:11214970 — UniProt-cited evidence.
- PubMed PMID:12168954 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ARHGAP35 (Rho GTPase-activating protein 35). Expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A03592 at 1:100-1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Breast (Glandular cells); negative Liver. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human brain tissue; fixative not specified (datasheet A03592); 2. Fixation: Image fixative and duration unreported (datasheet A03592); 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-ARHGAP35, 1:100-1:300 (datasheet A03592); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A03592); Caveat: Signaling may shift protein to membrane ruffles (UniProt); Isoform / epitope: No annotated isoforms; one intact chain (UniProt); Regulation: Expression regulation unreported (UniProt). Evidence: UniProt Q9NRY4, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nuclear bodies. In addition localized to the…, Datasheet A03592, PMC9342288. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ARHGAP35/immunohistochemistry-ihc-guide.