This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
RASAL3 / RAS protein activator like-3 · IHC design guide
Design Immunohistochemistry for RASAL3
Plan RASAL3 paraffin-section IHC around cytoplasmic staining in immune-cell subsets (HPA tissue IHC). Use lymph node non-germinal center cells as a high-staining reference (HPA tissue IHC) and start with the IHC-validated antibody at 2–5 μg/ml (datasheet A11713-1).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RASAL3 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 | Cytoplasm in immune-cell subsets (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in subsets of immune cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A11713-1) | |
| Positive control | Lymph node+2 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 paraffin sections (standard IHC practice; not target-specific) | |
| Caveat | Subset-restricted staining may be missed (HPA tissue IHC) | |
| Regulation | Predominant in hematopoietic lineages (UniProt) | |
| Isoform / epitope | 2 isoforms; confirm epitope coverage (UniProt) |
Section 1
Recommended RASAL3 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: A11713-1) is accompanied by a published lung adenocarcinoma tissue microarray protocol (PMC9753442).
| Sample | Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet A11713-1) |
| Fixation | Image fixative and duration unreported (datasheet A11713-1); 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 A11713-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A11713-1) |
| Primary antibody | Rabbit anti-RASAL3, 2-5 μg/ml (datasheet A11713-1) |
| Primary incubation | Overnight at 4 °C (datasheet A11713-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A11713-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | RASAL3-positive staining in non-germinal center cells of lymph node (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in subsets of immune cells. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet: A11713-1); the published protocol reports retrieval without specifying its conditions (PMC9753442).
Section 2
What Is the Expected RASAL3 Staining Pattern?
In paraffin section IHC, expect RASAL3 staining in the cytoplasm of subsets of immune cells, consistent with its cytoplasmic and cell cortex annotation and lack of a transmembrane segment (HPA tissue IHC; UniProt Q86YV0). HPA rates its tissue staining reliability as Enhanced, with medium consistency between staining and RNA data and external verification pending (HPA tissue IHC).
| Cytoplasmic staining in subsets of lymph node or tonsil non-germinal center cells, with less staining in spleen white pulp cells. | This fits the reported High staining in those lymph node and tonsil cells and Medium staining in spleen white pulp cells (HPA tissue IHC). Compare cell populations within the section; a uniformly stained tissue is a different pattern. |
| Predominantly nuclear staining, with little cytoplasmic staining, on a paraffin IHC slide. | Recheck localisation and controls before calling this the expected IHC result: HPA describes cytoplasmic tissue staining (HPA tissue IHC). Nucleoplasmic localisation is approved in HPA ICC-IF, so a nuclear IF image alone does not establish the expected paraffin IHC pattern (HPA subcellular). |
| Strong staining in adipocytes or glandular cells instead of the expected immune cell subsets. | Treat it as possible cross-reactivity or endogenous detection activity and inspect controls: HPA reports RASAL3 as Not detected in adipose tissue adipocytes and adrenal gland glandular cells (HPA tissue IHC). Cell identity matters more than a positive-looking field. |
| Diffuse colour across cells, stroma and the slide background. | A diffuse field does not reproduce the reported cytoplasmic staining in immune cell subsets (HPA tissue IHC). General IHC causes include insufficient blocking, inadequate washing or endogenous detection activity; compare a primary-antibody-omission control before assigning the colour to RASAL3. |
| No staining in lymph node non-germinal center cells on a test section. | That conflicts with the reported High staining in this population (HPA tissue IHC). Confirm the cell population and section quality, then check the antibody's IHC-P instructions, antigen retrieval and detection controls before interpreting other negative sections. |
💡Expected RASAL3 appearanceCall a paraffin IHC result positive when immune cell subsets show cytoplasmic staining, especially High staining in lymph node or tonsil non-germinal center cells; broad staining of HPA-negative cell types or diffuse background calls for controls (HPA tissue IHC).
| Tissue and cell selection | Use reported positive cell populations to judge the run: lymph node and tonsil non-germinal center cells stain High, while spleen white pulp cells stain Medium (HPA tissue IHC). An apparently empty field may simply miss the relevant cells. |
| RNA and protein readouts | Bone marrow belongs to an RNA-enriched tissue group, yet its hematopoietic cells are reported as Not detected by IHC (HPA tissue IHC). Do not substitute RNA enrichment for an observed IHC positive control. |
| Antibody evidence | The listed HPA antibody, HPA043417, has Enhanced IHC validation and Uncertain ICC validation; HPA also notes medium staining-to-RNA consistency and pending external verification (HPA antibodies; HPA tissue IHC). Interpret unexpected compartments with those limits in view. |
| Isoforms and epitope | UniProt lists 2 RASAL3 isoforms and PH, C2 and Ras-GAP domains (UniProt Q86YV0). No antibody epitope or isoform coverage is supplied, so the record cannot predict which isoform a particular stain detects. |
| IF/ICC Q: Should its compartment match paraffin IHC? | A: HPA ICC-IF reports approved nucleoplasmic and supported plasma membrane localisation, whereas HPA tissue IHC describes cytoplasmic staining in immune cell subsets (HPA subcellular; HPA tissue IHC). Judge each application against its own evidence. |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in a section chosen as a positive control | The field may lack the reported positive cell subset, or an IHC workflow step may have failed; lymph node non-germinal center cells are reported High (HPA tissue IHC). | Locate that cell population on the counterstained section. Check the catalog antibody's IHC-P instructions, the retrieval step and detection controls before adjusting the assay; the supplied record gives no target-specific retrieval condition. |
| Bone marrow gives little or no staining despite RNA enrichment | HPA reports bone marrow hematopoietic cells as Not detected by tissue IHC despite bone marrow's inclusion in an RNA-enriched group (HPA tissue IHC). | Use a lymph node or tonsil section containing non-germinal center cells to evaluate the IHC run (HPA tissue IHC). Record the bone marrow finding by cell type rather than treating its RNA category as an IHC control. |
| Colour appears in adipocytes or adrenal gland glandular cells | These cell types are reported Not detected (HPA tissue IHC); cross-reactivity or endogenous detection activity is possible. | Compare a primary-antibody-omission control and inspect whether colour follows cells or the detection system. Reassess blocking and antibody concentration using the catalog IHC-P instructions. |
| Most of the section has diffuse chromogenic background | Non-specific binding, residual detection reagent or endogenous activity can produce background in general IHC practice; HPA instead reports staining in immune cell subsets (HPA tissue IHC). | Check blocking, washes and the omission control. Score only interpretable cells after background is controlled; do not count uniform haze as cytoplasmic RASAL3 staining. |
| The strongest paraffin IHC signal appears nuclear | This differs from HPA's cytoplasmic tissue IHC profile, although nucleoplasmic localisation is approved in HPA ICC-IF (HPA tissue IHC; HPA subcellular). | Check compartment boundaries with the counterstain and compare a reported IHC-positive cell population and controls. Document the IHC observation separately from the ICC-IF localisation. |
| Two sections seem to disagree in staining intensity | HPA reports different levels across positive populations: High in lymph node and tonsil non-germinal center cells and Medium in spleen white pulp cells (HPA tissue IHC). | Compare the same identified cell type and compartment before judging assay performance. Note tissue and cell population alongside intensity; HPA's reliability statement also flags medium consistency with RNA (HPA tissue IHC). |
Experiment evidence
IHC & IF images showing RASAL3 staining
A11713-1 · IHCIHC analysis of RASAL3 using anti-RASAL3 antibody (A11713-1). RASAL3 was detected in a paraffin-embedded section of human lung cancer tissue
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000105122-RASAL3/tissue/cerebral+cortex#imid_14649004
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000105122-RASAL3/tissue/colon#imid_14648922
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000105122-RASAL3/tissue/liver#imid_14648931
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000105122-RASAL3/tissue/kidney#imid_14648924
HPA · ICC-IF HELHuman Protein Atlas immunofluorescence, HEL, antibody HPA043417; green = RASAL3, blue = nucleus, red = microtubules.
HPA · ICC-IF HELHuman Protein Atlas immunofluorescence, HEL, antibody HPA043417; green = RASAL3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RASAL3 IHC & IF
🧪Run lymph node first and look for staining in non-germinal center cells (HPA: High in lymph node non-germinal center cells). Use adipose tissue adipocytes as the negative comparison (HPA: Not detected in adipocytes); on the positive slide, cells outside the stained compartment should show only background, with their identity and staining assessed rather than assumed (HPA: lymph node staining reported for non-germinal center cells).
Positive control tissue: Lymph node (Non-germinal center cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RASAL3 in HEL, with annotated localisation: Nucleoplasm (approved), Plasma membrane (supported) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, a concentration-matched rabbit IgG isotype control, and a matched RASAL3 knockout specimen where available (caption: rabbit primary antibody; standard IHC practice). Quench endogenous peroxidase and check for DAB background in lymph node immune cells (caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: The selected A11713-1 paraffin-section caption reports heat retrieval in EDTA at pH 8.0, but its fixative is unreported; no target-specific fixation window or fixation effect is reported in the supplied evidence (caption: retrieval and fixative details). Treat retrieval as a condition to verify for this IHC assay, and check immune-cell peroxidase background when scoring lymph node (caption: HRP/DAB detection; standard IHC practice). The supplied evidence does not establish whether frozen sections or IF are easier for RASAL3 (caption: paraffin IHC; HPA: HEL ICC-IF images).
HPA tissue IHC evidence for RASAL3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — 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 |
|---|---|---|---|---|
| Lymph node | Non-germinal center cells | High | Protein (IHC) | HPA → |
| Tonsil | Non-germinal center cells | High | Protein (IHC) | HPA → |
| Spleen | Cells in white pulp | 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 → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RASAL3 IHC Tips
Troubleshoot RASAL3 staining in paraffin sections by checking retrieval, compartment, cell identity and controls before interpreting DAB signal.
Which retrieval condition should I try first for weak RASAL3 staining?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A11713-1). The selected RASAL3 tissue image used this condition, followed by 10% goat serum blocking and 2 μg/ml primary antibody overnight at 4°C (datasheet A11713-1). If staining is weak, check that sections were fully deparaffinised and that heating, cooling and washes were consistent across slides (standard IHC practice). Compare any adjusted retrieval condition on adjacent sections with the same positive control, because stronger DAB signal alone does not establish better specificity (standard IHC practice).
Could fixation explain inconsistent RASAL3 staining between paraffin blocks?
Target-specific sensitivity of RASAL3 to fixation is unknown: the selected paraffin-section caption does not report a fixative (datasheet A11713-1). Record the fixative, fixation duration and processing history for each block before comparing staining intensity (standard IHC practice). On matched sections, keep EDTA retrieval at pH 8.0 and the primary antibody at 2 μg/ml constant while assessing the effect of processing differences (datasheet A11713-1; standard IHC practice). Include a positive tissue control in each run, and avoid treating variation between blocks as a biological difference until cell composition and section quality have been checked (standard IHC practice).
How should I interpret cytoplasmic, cortical and nuclear RASAL3 staining?
Expect cytoplasmic signal, potentially enriched at the cell cortex, from the UniProt localisation annotation for RASAL3 (UniProt Q86YV0). Tissue IHC reports cytoplasmic expression in subsets of immune cells, whereas HPA ICC/IF lists nucleoplasm and plasma membrane, so assess compartments within the application used (HPA tissue IHC; HPA subcellular). RASAL3 has no annotated transmembrane segment, making a sharp extracellular or luminal pattern difficult to explain from its recorded topology (UniProt Q86YV0 topology). Compare suspicious nuclear or membrane staining with the counterstain, cell boundaries, positive tissue control and primary-omission control before assigning localisation (standard IHC practice).
Could isoforms or epitope accessibility cause discordant RASAL3 staining?
RASAL3 has 2 annotated isoforms, but the supplied antibody caption does not identify its epitope or establish which isoforms it detects (UniProt Q86YV0; datasheet A11713-1). Its PH, C2 and Ras-GAP domains occupy residues 197–293, 284–404 and 474–682, respectively (UniProt Q86YV0). Several phosphorylated residues are annotated, including serines 18 and 51; their effects on this antibody's staining are unknown (UniProt Q86YV0; datasheet A11713-1). If results disagree, obtain the antibody's immunogen or epitope information, then check isoform coverage and compare matched sections under identical retrieval conditions (standard IHC practice).
How can I check RASAL3 localisation by IF alongside tissue IHC?
For a separate IF/ICC experiment, multiplex RASAL3 with a validated immune cell marker to identify the stained population, since tissue IHC reports expression in subsets of immune cells (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores and place the weaker signal in a far-red channel when tissue autofluorescence compromises shorter wavelengths (standard IF practice). Permeabilise cells when testing cytoplasmic or nucleoplasmic epitopes, and optimise that step against membrane-boundary preservation if assessing cortical signal (UniProt Q86YV0; HPA subcellular; standard IF practice). HPA reports ICC/IF images in HEL cells, but its subcellular observations do not establish how the catalog antibody performs in IF (HPA subcellular; datasheet A11713-1).
What should I check when RASAL3 DAB staining appears diffuse?
Run primary-omission and secondary-only controls to locate reagent-derived background, and inspect a positive control alongside the test section (standard IHC practice). The selected caption used 10% goat serum blocking, a rabbit primary at 2 μg/ml, and an HRP-linked secondary before DAB development (datasheet A11713-1). For diffuse colour, review peroxidase blocking, wash quality, antibody concentration and DAB development across the run (standard IHC practice). Interpret staining in relation to cell boundaries and morphology: the reported tissue pattern is cytoplasmic in subsets of immune cells, so uniform staining across unrelated cells warrants further control checks (HPA tissue IHC; standard IHC practice).
How should I quantify RASAL3 staining in heterogeneous tissue? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because reported tissue staining is cytoplasmic in subsets of immune cells (HPA tissue IHC). An H-score can combine the percentage of cells at each intensity; report the percentage positive and, when cell abundance varies, positive-cell density per mm² as complementary measures (standard IHC practice). Normalise cell-level scores to the number of eligible cells and area-based density to evaluable tissue area, excluding folds and necrosis by a prespecified rule (standard IHC practice). Keep retrieval, imaging, DAB development and scoring thresholds consistent, and assess positive controls across runs before comparing specimens (standard IHC practice).
What distinguishes credible RASAL3 positivity from staining artefact?
Credible tissue IHC signal should be judged against the reported cytoplasmic staining in subsets of immune cells and the UniProt cytoplasmic or cortical annotation (HPA tissue IHC; UniProt Q86YV0). HPA reports high staining in non-germinal-center cells of lymph node and tonsil, but describes only medium agreement between staining and RNA data pending external verification (HPA tissue IHC). Check whether signal follows identifiable cells instead of section edges or necrotic areas, and whether it persists after appropriate endogenous peroxidase blocking (standard IHC practice). Use primary-omission controls and matched positive tissue to investigate unexpected nuclear, widespread epithelial or diffuse extracellular DAB signal before assigning RASAL3 expression (standard IHC practice).
Boster reagents
Best RASAL3 / RAS protein activator like-3 IHC Antibodies
The IHC-validated antibody has a figure from paraffin-embedded human lung cancer tissue (image caption). Human and mouse reactivity are listed, but the IHC dilution is specified for human samples (catalog).
Real IHC data
Anti-RASAL3 Antibody ®
Cat # A11713-1
A11713-1 is listed for IHC and has an IHC image from a paraffin-embedded human lung cancer section (catalog applications; image caption). The catalog lists human and mouse reactivity, while its IHC dilution is specified for human samples (catalog reactivity; catalog dilution).
Which to pick: Choose A11713-1 for human paraffin-section IHC; its caption documents EDTA retrieval at pH 8.0, overnight primary incubation at 2 μg/ml, and HRP–DAB detection, but does not report the fixative (image caption). Mouse reactivity is listed, though the supplied IHC dilution is specified for human samples (catalog reactivity; catalog dilution). There is no IF/ICC protocol choice here: IF/ICC is absent from the listed applications, and no IF image is supplied (catalog applications; catalog IF images).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q86YV0 (RASL3_HUMAN, RAS protein activator like-3).
- Human Protein Atlas. RASAL3 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. RASAL3 subcellular location (ICC-IF): Localized to the nucleoplasm and plasma membrane..
- Human Protein Atlas. RASAL3 antibody validation summary (1 antibodies).
- RASAL3 predicts overall survival and CD8+ T lymphocyte infiltration in lung adenocarcinoma. Journal of cellular and molecular medicine 2022 — PMC9753442.
- CCDC88B interacts with RASAL3 and ARHGEF2 and regulates dendritic cell function in neuroinflammation and colitis. Communications biology 2024 — PMC10781698.
- PubMed PMID:15057824 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RASAL3 (RAS protein activator like-3). Expected localisation Cytoplasm in immune-cell subsets (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A11713-1). Validated IHC antibody: A11713-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Lymph node (Non-germinal center cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet A11713-1); 2. Fixation: Image fixative and duration unreported (datasheet A11713-1); 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 A11713-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A11713-1); 8. Primary antibody: Rabbit anti-RASAL3, 2-5 μg/ml (datasheet A11713-1); 9. Primary incubation: Overnight at 4 °C (datasheet A11713-1). Watch for: Fixation: Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific); Caveat: Subset-restricted staining may be missed (HPA tissue IHC); Isoform / epitope: 2 isoforms; confirm epitope coverage (UniProt); Regulation: Predominant in hematopoietic lineages (UniProt). Evidence: UniProt Q86YV0, HPA tissue IHC · Enhanced, HPA ICC-IF · nucleoplasm and plasma membrane., Datasheet A11713-1, PMC9753442. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RASAL3/immunohistochemistry-ihc-guide.