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RPH3A / Rabphilin-3A · IHC design guide
Design Immunohistochemistry for RPH3A
Plan RPH3A paraffin-section IHC around strong cerebellar neuronal staining and moderate cortical neuropil staining (HPA tissue IHC). Use the guide to choose controls, optimize staining, and interpret regional differences.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RPH3A 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 | Neuronal cytoplasm/membrane and neuropil (HPA tissue IHC) | |
| Staining pattern | Cerebellar neurons: cytoplasm/membrane; cortical neuropil (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A10247-2) | |
| Positive control | Cerebellum+1 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep paraffin-section fixation consistent across samples. (standard IHC practice; not target-specific) | |
| Caveat | Staining varies by brain region; hippocampus is low (HPA tissue IHC) | |
| Regulation | Expression regulation not annotated (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage unspecified (UniProt) |
Section 1
Recommended RPH3A IHC & IF Protocols
The catalog antibody uses EDTA retrieval (datasheet A10247-2); the published RPH3A protocols below cover free-floating hippocampal slices (PMC10917878) and paraffin-embedded brain sections (PMC7923505).
| Sample | Paraffin-embedded mouse brain tissue; fixative not specified (datasheet A10247-2) |
| Fixation | Image fixative and duration unreported (datasheet A10247-2); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A10247-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A10247-2) |
| Primary antibody | Rabbit anti-RPH3A, 2-5 μg/ml (datasheet A10247-2) |
| Primary incubation | Overnight at 4 °C (datasheet A10247-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A10247-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | RPH3A-positive staining in gLUC cells - cytoplasm/membrane of cerebellum (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in neuronal cells and neuropil. No signal in the no-primary control. |
💡Decision noteFor paraffin sections, start with heat-mediated EDTA at pH 8.0 (datasheet A10247-2); citrate at pH 6.1 is a published alternative (PMC7923505).
Section 2
What Is the Expected RPH3A Staining Pattern?
RPH3A is associated with synaptic vesicle membranes, dendritic spines and postsynaptic membranes, with no transmembrane segment (UniProt Q9Y2J0: location, topology). In tissue IHC, expect cytoplasmic staining in neuronal cells and neuropil (HPA: tissue IHC profile). HPA rates the tissue staining evidence Enhanced, citing consistency between antibody staining and RNA expression (HPA: reliability). Interpret the pattern by cell type and compartment as well as intensity.
| Strong cytoplasmic/membrane staining in cerebellar GLUC cells. | This matches a reported positive tissue and cell population (HPA: cerebellum, GLUC cells, High). Assess the cellular pattern across the section; RPH3A's vesicle and membrane associations make cytoplasmic and membrane-associated signal plausible, without requiring a continuous cell-outline stain (UniProt Q9Y2J0: location, topology). |
| Moderate staining in cerebral cortex neuropil; weaker neuronal staining in hippocampus or caudate. | These relative levels follow the tissue observations: cortical neuropil is Medium, while hippocampal and caudate neuronal cells are Low (HPA: tissue IHC). A weaker field in either Low category is therefore less informative than a failed High cerebellar control (HPA: tissue IHC levels). |
| Predominantly nuclear staining, with little cytoplasmic or neuropil signal. | This conflicts with the reported tissue profile and listed cellular locations (HPA: cytoplasmic neuronal and neuropil expression; UniProt Q9Y2J0: location). Treat it as suspect staining and compare with a known-positive section and a negative detection control before assigning it to RPH3A (general IHC practice). |
| Prominent signal in adipocytes or in adrenal glandular cells. | Those specific populations are reported as Not detected (HPA: adipose adipocytes; adrenal glandular cells). First check morphology and controls; staining there may reflect nonspecific antibody binding or endogenous detection activity, so appearance alone does not establish RPH3A expression (general IHC practice). |
| Diffuse color across tissue or no signal in cerebellar GLUC cells. | Diffuse deposition lacks the reported neuronal and neuropil distribution, while absent staining in a High population undermines a negative call elsewhere (HPA: tissue IHC profile; cerebellum, High). Review negative controls and the positive control on the same staining run before interpreting test sections (general IHC practice). |
💡Expected RPH3A appearanceCall a section positive when neuronal cytoplasm/membrane and neuropil carry anatomically coherent signal, strongest in cerebellar GLUC cells and moderate in cortical neuropil (HPA: cerebellum High; cerebral cortex Medium); isolated nuclear staining or strong staining of HPA Not detected cell populations is suspect (HPA: tissue IHC profile and negatives; UniProt Q9Y2J0: location).
| Tissue and cell selection | Cerebellar GLUC cells provide a High reference, cortical neuropil a Medium reference, and hippocampal or caudate neuronal cells Low references (HPA: tissue IHC). Adipocytes and adrenal glandular cells are reported Not detected; use these observations to choose comparisons, while keeping cell identity visible during scoring (HPA: tissue IHC). |
| Antibody evidence | HPA assigns antibody HPA002475 Enhanced IHC status and reports consistency between antibody staining and RNA expression (HPA: antibody validation; tissue reliability). That supports the observed IHC distribution; it does not specify the epitope, establish performance for every catalog antibody, or validate an IF appearance (HPA: supplied antibody and subcellular records). |
| Isoforms and epitope coverage | UniProt lists two RPH3A isoforms and a full-length chain spanning residues 1–694 (UniProt Q9Y2J0: isoforms, processing). The supplied records do not map the IHC antibody epitope, so isoform-specific staining or loss of a particular isoform cannot be inferred from a positive or negative section (HPA: supplied antibody record). |
| Can IF/ICC confirm the same pattern? | HPA supplies no ICC/IF cell images or main subcellular location for RPH3A, and lists no ICC validation for HPA002475 (HPA: subcellular record; antibody record). UniProt's vesicle and membrane locations offer a localisation expectation, but there is no supplied IF image pattern to use as an acceptance criterion (UniProt Q9Y2J0: location). |
| Situation | Likely cause | Next action |
|---|---|---|
| No chromogenic signal in the cerebellar positive control. | A High reference population should be visible: cerebellar GLUC cells are reported High (HPA: tissue IHC). A failed control can reflect a staining-run or detection problem (general IHC practice). | Confirm cell identity and section integrity, then review antibody dilution, antigen retrieval, detection reagents and counterstain against the run record (general IHC practice). Repeat the run with the known-positive control before interpreting negative test sections (general IHC practice). |
| Only nuclei stain in an otherwise intact neuronal section. | Nuclear-only staining disagrees with the cytoplasmic neuronal and neuropil tissue profile and UniProt cellular locations (HPA: tissue IHC; UniProt Q9Y2J0: location). | Compare the same run's cerebellar positive and negative detection controls; check whether the nuclear color persists without primary antibody (general IHC practice). Score the nuclear-only pattern as unconfirmed until the expected compartment is recovered (HPA: tissue IHC profile). |
| Strong color appears in adipocytes or adrenal glandular cells. | These cells are listed as Not detected, so strong staining needs a specificity check (HPA: tissue IHC negatives). Nonspecific binding or endogenous detection activity are possible technical causes (general IHC practice). | Inspect a no-primary control and the distribution of color within the cells; verify blocking and detection steps, then compare with the cerebellar positive control (general IHC practice; HPA: cerebellum High). Avoid labeling the unexpected color RPH3A on morphology alone (HPA: tissue IHC negatives). |
| Diffuse color obscures neuronal cytoplasm and neuropil. | The recorded pattern is cell and region associated, rather than uniform color across tissue (HPA: tissue IHC profile). General IHC causes include excessive antibody or detection background and inadequate washing (general IHC practice). | Inspect the negative detection control, then adjust antibody dilution, washes or detection development using the catalog antibody's validated IHC instructions (general IHC practice). Preserve a cerebellar positive control to check that background reduction retains the expected High signal (HPA: cerebellum High). |
| Cortical neuropil is visible, but hippocampal or caudate neurons seem negative. | The reported levels differ: cortical neuropil is Medium and hippocampal and caudate neuronal cells are Low (HPA: tissue IHC). A weak Low population may be hard to judge when staining contrast is limited (general IHC practice). | Compare sections stained in the same run and verify the High cerebellar reference first (HPA: tissue IHC levels; general IHC practice). Record Low populations as below detection when appropriate, without treating that result as evidence of global RPH3A absence (HPA: hippocampus and caudate Low). |
| An IF/ICC image is requested as proof of the IHC result. | The supplied HPA subcellular record has no ICC/IF images or main location, and HPA002475 has no listed ICC validation (HPA: subcellular record; antibody record). | Use the tissue IHC pattern and its positive and negative controls for this IHC interpretation (HPA: tissue IHC; general IHC practice). Treat any IF/ICC localisation assessment as a separate experiment requiring its own validation; do not infer an IF acceptance pattern from these HPA records (HPA: subcellular record). |
Experiment evidence
IHC & IF images showing RPH3A staining
A10247-2 · IHCIHC analysis of RPH3A using anti-RPH3A antibody (A10247-2). RPH3A was detected in a paraffin-embedded section of mouse brain tissue. Heat me
A10247-2 · IHCIHC analysis of RPH3A using anti-RPH3A antibody (A10247-2). RPH3A was detected in a paraffin-embedded section of rat brain tissue. Heat medi
A10247-1 · IHCImmunohistochemistry (IHC) analyzes of Rabphilin-3A (P231) pAb in paraffin-embedded human breast carcinoma tissue at 1:50.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000089169-RPH3A/tissue/cerebral+cortex#imid_1010720
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000089169-RPH3A/tissue/colon#imid_1010654
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000089169-RPH3A/tissue/liver#imid_1010647
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000089169-RPH3A/tissue/kidney#imid_1010656
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RPH3A IHC & IF
🧪Run cerebellum first and look for cytoplasmic and membranous staining in GLUC cells (HPA: High in cerebellar GLUC cells). Use adipose tissue as a negative and expect adipocytes to remain unstained (HPA: Not detected in adipocytes); on the cerebellar slide, cells outside the GLUC-cell staining pattern should show only background signal.
Positive control tissue: Cerebellum (GLUC cells - cytoplasm/membrane, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for RPH3A; derive a cell-line control from the positive tissue's cell type (GLUC cells - cytoplasm/membrane) and confirm it by RNA or western blot first.
Technical controls: Include no-primary (secondary-only) and rabbit IgG isotype controls matched to the primary antibody's clonality and concentration, plus RPH3A-knockout tissue or a peptide-block control if available (caption: rabbit primary; standard IHC practice). Block endogenous peroxidase and compare cerebellar DAB background with the no-primary control (caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A10247-2 paraffin-section caption does not state the fixative (selected-SKU caption). The caption reports heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required (selected-SKU caption). Frozen-section performance and whether IF is easier are unreported; in cerebellum, background DAB signal could obscure the expected GLUC-cell cytoplasmic and membranous pattern, so assess it against the no-primary control (HPA: High in cerebellar GLUC cells; standard IHC practice).
HPA tissue IHC evidence for RPH3A
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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 | GLUC cells - cytoplasm/membrane | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuropil | 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 → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RPH3A IHC Tips
Troubleshoot RPH3A staining in paraffin sections by checking retrieval, compartment, controls and scoring before interpreting chromogenic signal.
Which retrieval conditions should I start with for RPH3A in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A10247-2). The catalog antibody produced RPH3A staining in mouse brain after this retrieval, although the caption gives no heating time or temperature (datasheet A10247-2). If staining is weak, compare shorter and longer heating within your validated retrieval workflow while holding antibody concentration and detection constant (standard IHC practice). Record tissue preservation alongside signal, because excessive heating can damage section morphology and complicate interpretation (standard IHC practice). Evaluate the expected neuronal cytoplasmic and neuropil pattern before accepting a stronger stain as an improvement (HPA tissue IHC).
Could fixation be responsible for weak or uneven RPH3A staining?
Target-specific fixation sensitivity is unknown: the selected mouse brain caption identifies a paraffin section but does not state its fixative (datasheet A10247-2). Record the fixative, fixation duration, tissue thickness and processing history for each block before comparing staining (standard IHC practice). If those factors vary, stain matched sections together with the same pH 8.0 EDTA retrieval and detection settings (datasheet A10247-2; standard IHC practice). Compare staining with tissue morphology and an appropriately processed positive control, because poor preservation can make local signal hard to assess (standard IHC practice). Do not assign a fixation effect to RPH3A without a direct comparison using controlled samples (standard IHC practice).
Where should chromogenic RPH3A signal appear in brain sections?
Look for neuronal cytoplasmic and neuropil staining, which matches the reported human tissue pattern (HPA tissue IHC). RPH3A is associated with cytoplasmic secretory and synaptic vesicles, dendritic spines and postsynaptic membranes, without a transmembrane segment (UniProt Q9Y2J0 localisation and topology). Therefore, a punctate or process-associated pattern can be plausible, but diffuse chromogen alone does not establish vesicle localisation (UniProt Q9Y2J0 localisation; standard IHC interpretation). Use the tissue architecture and a neuronal counterstain or adjacent reference section to distinguish cell bodies from neuropil (standard IHC practice). Treat strong nuclear-only staining as discordant with the supplied localisation and check controls before scoring it (HPA tissue IHC; UniProt Q9Y2J0 localisation).
How can an unknown epitope affect interpretation across RPH3A isoforms?
RPH3A has 2 reported isoforms, while the supplied antibody caption does not identify its epitope or demonstrate isoform-specific recognition (UniProt Q9Y2J0 isoforms; datasheet A10247-2). Check the catalog antibody's documented immunogen or epitope against each isoform sequence before claiming isoform coverage (standard IHC practice). The protein contains a Rab-binding region at residues 44–160 and C2 domains at 392–514 and 550–683, so epitope position could affect which features are sampled (UniProt Q9Y2J0 domains). Report staining as RPH3A immunoreactivity unless specificity for a particular isoform has been established independently (standard IHC interpretation). If results differ between specimens, check section processing and controls before attributing the difference to alternative splicing (UniProt Q9Y2J0 isoforms; standard IHC practice).
How should I adapt RPH3A localisation questions to a multiplex IF experiment?
For a separate IF experiment, pair RPH3A with a marker of neuronal cells and assess whether signal follows neuronal processes or neuropil (HPA tissue IHC; standard IF practice). Choose fluorophores in channels with low measured tissue autofluorescence, and include single-label controls to assess spectral spillover (standard IF practice). RPH3A lacks a transmembrane segment and associates with vesicles and postsynaptic structures, so select permeabilisation according to the antibody epitope's accessibility on the relevant membrane side (UniProt Q9Y2J0 topology and localisation; standard IF practice). Because the supplied antibody evidence documents paraffin-section chromogenic staining, establish IF conditions and specificity independently rather than treating that caption as IF validation (datasheet A10247-2; standard IF practice).
What should I check when RPH3A DAB staining looks widespread?
Compare the suspect section with a no-primary control to identify signal from the detection system or endogenous peroxidase (standard IHC practice). Include a peroxidase block before HRP and DAB development, then keep development time consistent across the comparison set (standard IHC practice). The selected caption used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and DAB detection (datasheet A10247-2). If background persists, titrate primary antibody and shorten chromogen development while retaining a positive brain control (standard IHC practice; HPA tissue IHC). Widespread staining in compartments outside the reported neuronal cytoplasm and neuropil pattern warrants caution, particularly when controls also stain (HPA tissue IHC; standard IHC interpretation).
How should I score RPH3A when both neurons and neuropil stain? ⚠ ANSWER MARKED FOR VERIFICATION
Define neuronal cytoplasm and neuropil as separate regions before scoring, because both contribute to the reported tissue pattern (HPA tissue IHC). For neuronal cell bodies, record the percentage of positive cells and an H-score from prespecified intensity bins; retain the denominator of all evaluable neurons (standard IHC quantification). For neuropil, measure DAB-positive area or optical density per mm² of evaluable neuropil, using consistent thresholds and illumination (standard IHC quantification). Normalise each measurement to its annotated tissue compartment and exclude folds, torn edges and nonviable areas by a fixed rule (standard IHC quantification). Compare like anatomical regions and matched processing batches before interpreting differences in RPH3A abundance (standard IHC practice).
How can I distinguish genuine RPH3A staining from artefact?
A convincing result follows the neuronal cytoplasmic or neuropil pattern and remains absent from the corresponding no-primary control (HPA tissue IHC; standard IHC practice). HPA reports high staining in cerebellar GLUC cell cytoplasm or membrane and medium staining in cerebral cortex neuropil, providing region-specific reference patterns (HPA tissue IHC). Question nuclear-only or broadly glandular staining because it conflicts with the supplied localisation and tissue profile (UniProt Q9Y2J0 localisation; HPA tissue IHC). Exclude tissue edges, necrotic areas and deposits that track section damage rather than intact cellular structures (standard IHC interpretation). Check whether a peroxidase block removes suspect DAB signal before assigning it to RPH3A (standard IHC practice).
Boster reagents
Best RPH3A / Rabphilin-3A IHC Antibodies
Two anti-RPH3A antibodies have real paraffin-section IHC images from mouse and rat brain and human breast carcinoma (catalog IHC captions). IF is listed for A10247-1, without an IF image (catalog A10247-1).
Real IHC data
Anti-RPH3A Antibody ®
Cat # A10247-2
Real IHC data
Anti-Rabphilin-3A (P231) RPH3A Antibody
Cat # A10247-1
A10247-2 will render with mouse brain IHC; its catalog also documents rat brain IHC and lists human, mouse and rat reactivity (catalog A10247-2 IHC captions and reactivity). A10247-1 will render with human breast carcinoma IHC and lists IF plus human, mouse and rat reactivity (catalog A10247-1 IHC caption, applications and reactivity).
Which to pick: For paraffin-section tissue IHC, choose A10247-2 when the sample is mouse or rat brain: its own captions document EDTA pH 8 retrieval and DAB detection, with fixation unreported (catalog A10247-2 IHC captions). Choose A10247-1 for the pictured human breast carcinoma IHC example or as the IF candidate; its caption reports paraffin embedding but no fixative, and ICC is not listed (catalog A10247-1 IHC caption and applications). For cross-species planning, both list human, mouse and rat reactivity, while their pictured IHC evidence covers different species (catalog reactivity and IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9Y2J0 (RP3A_HUMAN, Rabphilin-3A).
- Human Protein Atlas. RPH3A tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. RPH3A subcellular location (ICC-IF): Highest expression in SH-SY5Y: 15.7 nTPM.
- Human Protein Atlas. RPH3A antibody validation summary (1 antibodies).
- Immunohistochemical field parcellation of the human hippocampus along its antero-posterior axis. Brain structure & function 2024 — PMC10917878.
- Association of CSF proteins with tau and amyloid β levels in asymptomatic 70-year-olds. Alzheimer's research & therapy 2021 — PMC7923505.
- Rabphilin3A reduces integrin-dependent growth cone signaling to restrict axon regeneration after trauma. Experimental neurology 2022 — PMC9555232.
- PubMed PMID:10231032 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RPH3A (Rabphilin-3A). Expected localisation Neuronal cytoplasm/membrane and neuropil (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A10247-2). Validated IHC antibody: A10247-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Cerebellum (GLUC 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 A10247-2); 2. Fixation: Image fixative and duration unreported (datasheet A10247-2); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: EDTA pH 8.0 (datasheet A10247-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A10247-2); 8. Primary antibody: Rabbit anti-RPH3A, 2-5 μg/ml (datasheet A10247-2); 9. Primary incubation: Overnight at 4 °C (datasheet A10247-2). Watch for: Fixation: Keep paraffin-section fixation consistent across samples. (standard IHC practice; not target-specific); Caveat: Staining varies by brain region; hippocampus is low (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage unspecified (UniProt); Regulation: Expression regulation not annotated (UniProt). Evidence: UniProt Q9Y2J0, HPA tissue IHC · Enhanced, HPA cell-line RNA · Highest expression in SH-SY5Y: 15.7 nTPM, Datasheet A10247-2, PMC10917878, PMC7923505. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RPH3A/immunohistochemistry-ihc-guide.