This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
ARHGAP35 / Rho GTPase-activating protein 35 · IHC design guide
Design Immunohistochemistry for ARHGAP35
Plan chromogenic ARHGAP35 IHC in paraffin sections around the cytoplasmic pattern reported in most tissues (HPA tissue IHC). Breast glandular cells show high staining, while liver cholangiocytes are reported as undetected; use these observations to guide controls and scoring (HPA tissue IHC).
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 in tissue IHC (HPA tissue IHC); nuclear and membrane locations are annotated (UniProt) | |
| Staining pattern | Cytoplasmic in most tissues; high in breast glandular cells (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 | Keep paraffin-section fixation consistent across samples. (standard IHC practice; not target-specific) | |
| Caveat | Signaling may shift ARHGAP35 toward membrane ruffles (UniProt) | |
| Regulation | Expression regulation not established (UniProt) | |
| Isoform / epitope | No listed isoforms or processing; map the epitope within residues 1–1499 (UniProt) |
Section 1
Recommended ARHGAP35 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is paired with a published gastric cancer tissue microarray method (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 retrieval at pH 9.0, 95–98 °C for 20 min (page antigen retrieval).
Section 2
What Is the Expected ARHGAP35 Staining Pattern?
ARHGAP35 is expected mainly in the cytoplasm of cells across many tissues (HPA: tissue IHC profile). High staining is reported in breast and endometrial glandular cells, cerebellar Purkinje cells, and cerebral cortical neurons (HPA: High in each). UniProt also lists cytoskeletal, nuclear, and cell membrane locations (UniProt Q9NRY4: subcellular location). It has no transmembrane segment (UniProt Q9NRY4: topology). HPA rates its tissue IHC profile Approved, with medium staining–RNA consistency and external verification pending (HPA: reliability).
| Cytoplasmic chromogen in breast or endometrial glandular cells, Purkinje cells, or cortical neurons (HPA: High in each). | This fits the reported tissue IHC profile (HPA: cytoplasmic expression in most tissues). Compare intensity within the relevant cell population; a positive tissue section establishes that the staining run worked, but HPA's Approved rating still carries medium staining–RNA consistency and pending external verification (HPA: reliability). |
| Predominantly nuclear chromogen, with little cytoplasmic staining, in a tissue section (HPA: tissue IHC profile). | Treat a nuclear-only IHC result as discordant with HPA's mainly cytoplasmic tissue pattern and review controls and morphology (HPA: tissue IHC profile; standard IHC practice). Do not call every nuclear signal artefactual: UniProt lists the nucleus, and HPA reports approved nuclear-body localisation by ICC-IF (UniProt Q9NRY4: subcellular location; HPA: ICC-IF). |
| Strong cholangiocyte staining in liver, especially if the no-primary control is also coloured (HPA: cholangiocytes Not detected). | The cell-type result conflicts with HPA's liver observation (HPA: cholangiocytes Not detected). Shared colour in a no-primary control suggests endogenous detection activity; colour confined to the primary-antibody section warrants checking cross-reactivity and scoring (standard IHC practice). One discordant section alone cannot identify the cause. |
| Diffuse colour over stroma, lumina, and several cell types without clear cellular boundaries (standard IHC practice). | This is difficult to score as ARHGAP35 because the expected tissue pattern identifies stained cells and their cytoplasm (HPA: tissue IHC profile). Review no-primary and tissue controls, then assess blocking, washing, and chromogen development as general IHC variables (standard IHC practice). |
| No chromogen in the expected cells of a breast or cerebellum section (HPA: High in breast glandular and cerebellar Purkinje cells). | An absent signal in a reported high-staining cell population raises a run or detection question, provided that population is present on the section (HPA: High in those cells; standard IHC practice). Check section identity, tissue preservation, antibody incubation, detection reagents, and counterstain before interpreting the sample as negative (standard IHC practice). |
💡Expected ARHGAP35 appearanceA convincing positive shows High cytoplasmic staining in the named glandular or neuronal cells (HPA: tissue IHC), while strong cholangiocyte colour or cell-independent diffuse colour is suspect (HPA: cholangiocytes Not detected; standard IHC practice).
| Choice of comparison tissue (HPA: tissue IHC). | Breast glandular cells, Purkinje cells, and cortical neurons are reported High; liver cholangiocytes are Not detected (HPA: tissue IHC). Compare like cell types when judging a run, since HPA reports low tissue specificity overall (HPA: RNA specificity). |
| Membrane-associated redistribution (UniProt Q9NRY4: subcellular location). | After integrin and SDC4 responses with PKC phosphorylation, ARHGAP35 can move from cytoplasm to plasma membrane ruffles and colocalise with polymerised actin (UniProt Q9NRY4: subcellular location). Focal peripheral staining can therefore merit closer review alongside the expected cytoplasmic tissue pattern (HPA: tissue IHC). |
| Ciliary staining (HPA: tissue IHC). | HPA reports High staining at cilia tips in fallopian tube and nasopharyngeal ciliated cells (HPA: tissue IHC). Assess those tips separately from broad surface colour when the relevant ciliated cells are present (HPA: tissue IHC; standard IHC practice). |
| Antibody evidence (HPA: antibody validation). | HPA055184, HPA056470, and CAB037311 each have Approved IHC status (HPA: antibody validation). The overall tissue profile still has medium staining–RNA consistency and awaits external verification (HPA: reliability); approval does not settle a discordant result in a particular section. |
| IF/ICC Q: Should its localisation match chromogenic tissue IHC? | A: HPA reports mainly nuclear bodies, plus microtubules and aggresomes, in ICC-IF; these locations are Approved (HPA: ICC-IF). Evaluate an IF image against that evidence while evaluating a paraffin tissue section against HPA's mainly cytoplasmic IHC profile (HPA: tissue IHC). |
| Protein topology and processing (UniProt Q9NRY4: topology and processing). | UniProt lists no transmembrane segment, signal peptide, or propeptide, and annotates one chain spanning residues 1–1499 (UniProt Q9NRY4: topology and processing). Those annotations alone do not predict antigen retrieval performance or a fixation-sensitive staining change. |
| Situation | Likely cause | Next action |
|---|---|---|
| Reported high-staining cells remain blank (HPA: breast glandular or cerebellar Purkinje cells, High). | The expected population may be absent, or an IHC run step may have failed (standard IHC practice). HPA's high rating describes observed staining; it does not identify which run step failed (HPA: tissue IHC). | Confirm the cell population on the counterstained section and inspect a positive control processed with the same run (standard IHC practice). Then check primary-antibody application, detection reagents, and chromogen development (standard IHC practice). |
| Liver cholangiocytes stain strongly (HPA: cholangiocytes Not detected). | The result is discordant with HPA; cross-reactivity or endogenous chromogen-generating activity are possible explanations (HPA: liver IHC; standard IHC practice). | Check a no-primary control on comparable liver tissue, confirm cell identity, and review detection blocking (standard IHC practice). If colour remains primary-dependent, treat the cell-type result as unresolved rather than assigning it to ARHGAP35. |
| Colour covers the section without distinct cytoplasmic cell outlines (HPA: cytoplasmic tissue profile). | Nonspecific binding, inadequate washing, or excess chromogen development can obscure cellular interpretation (standard IHC practice). This appearance alone does not establish ARHGAP35 expression. | Compare no-primary and positive-tissue controls; review blocking, washes, antibody incubation, and chromogen development within the general IHC workflow (standard IHC practice). Score only signal that can be assigned to cells and compartments. |
| Signal is mainly nuclear in paraffin tissue IHC (HPA: mainly cytoplasmic tissue profile). | The IHC pattern is discordant with HPA tissue images, although nuclear localisation is listed by UniProt and nuclear bodies are approved by HPA ICC-IF (UniProt Q9NRY4: subcellular location; HPA: ICC-IF). | Check tissue morphology, no-primary control, and cytoplasmic staining in a reported high-staining population (standard IHC practice; HPA: tissue IHC). Record the nuclear result as discordant until supported by controls. |
| A ciliated section shows only broad surface colour, with no discernible cilia tips (HPA: fallopian tube and nasopharynx IHC). | The reported cilia-tip pattern may be unresolved because the ciliated border is poorly preserved or visually indistinct (HPA: cilia-tip staining; standard IHC practice). | Verify that intact ciliated cells are visible with the counterstain, then assess tip-localised colour at suitable magnification (standard IHC practice). Do not score a feature that cannot be resolved. |
| A low-staining tissue looks weaker than a high-staining comparison (HPA: tissue IHC). | HPA reports Low staining in parathyroid glandular cells, ovarian follicle cells, skeletal myocytes, and splenic white-pulp cells, versus High in selected glandular and neuronal cells (HPA: tissue IHC). | Score the named cell type against its own reported level and retain a high-staining control in the run (HPA: tissue IHC; standard IHC practice). Weak colour in a Low category is not, by itself, evidence of assay failure. |
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: glandular cells should stain (HPA: High in breast glandular cells). Use liver cholangiocytes as the negative tissue (HPA: Not detected in liver cholangiocytes); on the breast slide, cells identified as unstained should show only background, but the supplied HPA rows do not establish a specific 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 antibody matched to the primary antibody’s host species and isotype; and a peptide-blocked section as a biological specificity control (A03592 caption: staining was blocked with synthesized peptide). For chromogenic breast IHC, check and quench endogenous peroxidase, and block endogenous biotin if using avidin–biotin detection (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported; the paraffin-section caption does not state the fixative (A03592 caption). ARHGAP35-specific antigen-retrieval dependence is unreported, so compare retrieval conditions on positive and negative sections (HPA: High in breast glandular cells; HPA: Not detected in liver cholangiocytes). An advantage for frozen sections or IF over paraffin IHC is unreported; assess background from endogenous peroxidase in breast sections (standard chromogenic IHC practice).
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 interpreting chromogenic signal.
How should I retrieve ARHGAP35 in paraffin sections when staining is weak?
Start with Tris-EDTA at pH 9.0, heated to 95–98 °C for 20 min (page retrieval setting: nuclear antigen). Allow sections to cool in the retrieval solution, then compare a known positive cell population and a no-primary control on the same run; breast glandular cells and cerebellar Purkinje cells are reported as high-staining populations (HPA tissue IHC). If signal remains weak, test a shorter heating time before changing buffer, because excessive retrieval can damage morphology and complicate compartment scoring (standard IHC practice). Record retrieval time, temperature, buffer, and section condition alongside each result so an apparent change in ARHGAP35 staining is not mistaken for a biological difference.
Could fixation explain variable ARHGAP35 staining between paraffin blocks?
ARHGAP35-specific fixation sensitivity is unknown from the supplied evidence; the selected image describes paraffin-embedded human brain tissue but does not report a fixative (A03592 tissue-IHC caption). Compare blocks with documented fixation duration and processing history, and keep section thickness, retrieval, and detection conditions consistent when testing a fixation hypothesis (standard IHC practice). If material permits, stain adjacent sections from differently processed blocks in the same run, using an internal positive cell population and a no-primary control to separate loss of antigen signal from background. Do not attribute a weak result to a particular fixative without a controlled comparison; the tissue staining pattern alone does not establish that cause.
Which cellular compartment should count as ARHGAP35-positive in chromogenic IHC?
Score cytoplasmic staining as the principal tissue pattern, while documenting nuclear or peripheral staining separately (HPA tissue IHC: cytoplasmic expression in most tissues; UniProt Q9NRY4: cytoplasm, nucleus, and cell membrane). ARHGAP35 can relocalize to actin-rich membrane ruffles following integrin and SDC4 signaling and PKC phosphorylation, so a peripheral pattern needs cellular context rather than automatic rejection (UniProt Q9NRY4 subcellular annotation). Nuclear bodies, microtubules, and aggresomes are reported in cell imaging, but that evidence does not establish how those structures appear in chromogenic tissue sections (HPA subcellular). Use a nuclear counterstain and intact morphology to assign signal to individual cells, and report compartment-specific scores instead of combining every brown deposit into one value.
How can an unknown antibody epitope affect ARHGAP35 IHC interpretation?
The supplied record lists 0 annotated isoforms and a 1–1499 amino-acid chain, but it does not locate the A03592 epitope (UniProt Q9NRY4 record; A03592 tissue-IHC caption). Consequently, do not assign a compartment-specific stain to a particular isoform or phosphorylation state without epitope mapping and independent validation; the record lists multiple modified residues, including phosphotyrosine 308 (UniProt Q9NRY4 modified residues). The selected brain image includes synthesized-peptide blocking, which supports competition for that image's signal but does not establish recognition of every tissue staining pattern (A03592 tissue-IHC caption). Compare staining with a second independently validated epitope when available, and document any disagreement by cell type and compartment rather than merging the scores.
How should I investigate a disputed ARHGAP35 pattern by IF?
Use IF as a separate validation experiment and multiplex ARHGAP35 with a marker identifying the expected cell type; for example, choose a neuronal marker when examining cerebral cortex neuronal cells (HPA tissue IHC: high in cerebral cortex neuronal cells). Select fluorophores after inspecting unstained tissue for autofluorescence, and favor a spectrally separated channel with low background for the weaker signal (standard IF practice). Because ARHGAP35 has no transmembrane segment and is annotated in intracellular compartments, include a controlled permeabilisation step when testing cytoplasmic or nuclear signal, while checking that morphology remains intact (UniProt Q9NRY4 topology and subcellular annotation; standard IF practice). Compare single-stain and no-primary controls before treating colocalisation as evidence of cell identity.
How do I distinguish ARHGAP35 staining from chromogenic background?
Inspect a no-primary section, an isotype control where appropriate, and tissue edges before judging diffuse brown staining; edge concentration and deposits unrelated to cell morphology warrant caution (standard IHC practice). Apply a peroxidase block before chromogenic detection and assess whether residual endogenous enzyme contributes to DAB signal, especially when a no-primary control is positive (standard chromogenic IHC practice). Titrate the catalog antibody and detection reagent while keeping retrieval fixed, then compare background with a reported positive population such as breast glandular cells (HPA tissue IHC: high in breast glandular cells). The peptide-blocked brain image provides a useful competition control for that specimen, but it does not replace controls on the tissue being scored (A03592 tissue-IHC caption).
What is a defensible way to quantify ARHGAP35 chromogenic staining? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because cytoplasmic tissue expression is commonly reported while nuclear and membrane locations also appear in the protein annotation (HPA tissue IHC; UniProt Q9NRY4 subcellular annotation). For intact cell populations, record the percentage of positive cells and an H-score using intensity categories 0–3; for discrete structures, report positive density per mm² (standard IHC scoring practice). Normalize counts to the number of evaluable cells of the same type or to annotated viable tissue area, and exclude folds, damaged edges, and necrotic regions by a prespecified rule. Keep imaging, threshold, counterstain, and observer criteria constant across sections, and report cytoplasmic and nuclear results separately.
When is an apparent ARHGAP35-positive cell or compartment convincing?
A convincing result follows intact cell morphology, appears in the expected compartment, and survives comparison with a no-primary control and relevant positive tissue (standard IHC practice; HPA tissue IHC: cytoplasmic expression in most tissues). Breast glandular cells and cerebellar Purkinje cells have reported high staining, whereas liver cholangiocytes are listed as not detected; treat these as reference patterns, not absolute controls for every specimen (HPA tissue IHC). Investigate staining confined to cut edges, necrotic areas, or cells with endogenous peroxidase activity before calling it ARHGAP35-positive (standard chromogenic IHC practice). Nuclear signal is biologically plausible, but unexpected nuclear-only staining should be documented and checked with an independent antibody or orthogonal assay (UniProt Q9NRY4 subcellular annotation; standard IHC validation practice).
Boster reagents
Best ARHGAP35 / Rho GTPase-activating protein 35 IHC Antibodies
Both antibodies have paraffin-embedded human brain IHC images (catalog image captions). IF is listed for both, ICC for P30433, and catalog reactivity covers human, mouse, and rat (catalog applications/reactivity).
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 brain paraffin-section IHC image with a peptide-blocked comparison; IF and human, mouse, and rat reactivity are listed (A03592 image caption; catalog applications/reactivity). P30433 has human brain and breast carcinoma paraffin-section IHC images with peptide-blocked comparisons; IF, ICC, and human, mouse, and rat reactivity are listed (P30433 image captions; catalog applications/reactivity).
Which to pick: For tissue IHC, choose A03592 for ARHGAP35 or P30433 for phospho-Tyr1105; both have human brain paraffin-section images, and P30433 also has a human breast carcinoma image (catalog titles; respective IHC image captions). For IF, both are listed; for ICC, choose P30433, the only ICC-listed option and one identified as polyclonal; A03592 clonality is unreported (catalog applications; P30433 dilution data; A03592 clone field). For cross-species work, both list human, mouse, and rat reactivity, although their IHC images show human tissue only; neither paraffin-section caption reports the fixative (catalog reactivity; 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 in tissue IHC (HPA tissue IHC); nuclear and membrane locations are annotated (UniProt). 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: Keep paraffin-section fixation consistent across samples. (standard IHC practice; not target-specific); Caveat: Signaling may shift ARHGAP35 toward membrane ruffles (UniProt); Isoform / epitope: No listed isoforms or processing; map the epitope within residues 1–1499 (UniProt); Regulation: Expression regulation not established (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.