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RAB27A / Ras-related protein Rab-27A · IHC design guide
Design Immunohistochemistry for RAB27A
Plan chromogenic RAB27A IHC on paraffin sections with the catalog antibody at 0.5–1 μg/mL (datasheet: A01608-1). Compare cytoplasmic staining in glandular cells of stomach and colon with the reported absence of staining in cervical glandular cells (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RAB27A 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 in most tissues, including immune cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet A01608-1) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
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 A01608-1) | |
| Caveat | Glandular staining varies; cervix is undetected (HPA tissue IHC) | |
| Regulation | Low expression in kidney and muscle (UniProt) | |
| Isoform / epitope | Long and Short isoforms; no transmembrane segment; epitope coverage unknown (UniProt) |
Section 1
Recommended RAB27A IHC & IF Protocols
Start with the catalog antibody’s IHC-P protocol (datasheet A01608-1), then compare the published RAB27A IHC methods below (PMC3935941; PMC13251207; PMC10079387).
| Sample | Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet A01608-1) |
| Fixation | Image fixative and duration unreported (datasheet A01608-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet A01608-1) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A01608-1) |
| Primary antibody | Rabbit anti-RAB27A, 0.5-1μg/ml (datasheet A01608-1) |
| Primary incubation | Overnight at 4 °C (datasheet A01608-1) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A01608-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | RAB27A-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues, including immune cells. No signal in the no-primary control. |
💡Decision noteTry citrate pH 6 heat retrieval first (datasheet A01608-1); EDTA pH 8 autoclave retrieval is a published alternative (PMC3935941).
Section 2
What Is the Expected RAB27A Staining Pattern?
RAB27A is a membrane-associated trafficking protein found on melanosomes, late endosomes, lysosomes and endosomal exocytic vesicles, without a transmembrane segment (UniProt P51159). In paraffin-section IHC, expect chiefly cytoplasmic staining in glandular cells of several digestive tissues and in some immune cells (HPA: tissue IHC profile). HPA rates its tissue staining reliability Enhanced, with medium consistency between antibody staining and RNA expression (HPA: reliability description).
| Cytoplasmic staining is clear in appendix, colon, duodenum, rectum, small-intestine or stomach glandular cells. | This matches HPA's High staining in those specified cell populations (HPA: tissue IHC). Vesicle-associated, sometimes granular cytoplasmic signal is biologically plausible given RAB27A's membrane-trafficking locations (UniProt P51159). Score the named cells and their cytoplasmic compartment; a whole-tissue label can hide differences between cell populations (general IHC interpretation). |
| Signal is predominantly nuclear, or forms a sharp continuous outline around every cell. | Question compartment assignment and staining specificity: HPA describes cytoplasmic tissue expression, while UniProt places RAB27A on intracellular membranes and vesicles and reports no transmembrane segment (HPA: tissue IHC profile; UniProt P51159). Inspect morphology and a matched control before treating an unexpected compartment as RAB27A (general IHC practice). |
| Strong staining appears chiefly in adipocytes, bronchial respiratory epithelium or esophageal squamous epithelium. | Those specified cell populations are Not detected in HPA tissue IHC, so strong signal there is discordant with the reference pattern (HPA: tissue IHC). Consider cross-reactivity or endogenous detection activity; check the antibody and detection controls before concluding that the sample has an altered expression pattern (general IHC practice). |
| Chromogen appears diffusely across the section, including areas without a coherent cell-associated pattern. | Uniform haze obscures the expected cytoplasmic cell pattern (HPA: tissue IHC profile). Possible general IHC causes include inadequate blocking, excessive detection reagent or incomplete washing (general IHC practice). Compare a no-primary control and local tissue morphology; background alone cannot establish RAB27A expression (general IHC practice). |
| A matched known-positive glandular region has no detectable staining. | HPA reports High glandular-cell staining in stomach and several intestinal sites, making those specified cells useful reference regions (HPA: tissue IHC). A blank reference region raises a run-performance or specimen-quality question; review the same-run control, antibody use, retrieval and detection steps before interpreting the test region as negative (general IHC practice). |
💡Expected RAB27A appearanceCall positive when glandular-cell cytoplasm shows clear, cell-associated chromogenic staining, potentially granular with vesicles (HPA: High in specified glandular cells; UniProt P51159 localisation); predominant nuclear signal or equally strong staining in HPA Not detected cell populations is suspect (HPA: tissue IHC).
| Which tissue and cell population is being read? | HPA reports High glandular-cell staining in appendix, colon, duodenum, prostate, rectum, small intestine and stomach, and Medium staining in bone-marrow hematopoietic cells (HPA: tissue IHC). It reports Not detected for particular populations, including adipocytes and glomerular cells; those calls do not make every cell in their tissues a negative control (HPA: tissue IHC). |
| What does antibody validation establish? | Two listed antibodies, HPA001333 and CAB034046, have Enhanced IHC status (HPA: antibody validation). HPA describes medium consistency between antibody staining and RNA expression overall (HPA: reliability description). Use that evidence to support the reported tissue pattern while assessing any new antibody or unexpected sample on its own merits (general IHC practice). |
| Do protein features change the predicted compartment? | UniProt lists long and short isoforms, no signal peptide or propeptide, and no transmembrane segment (UniProt P51159). Its membrane and vesicle localisation supports intracellular cytoplasmic signal; the supplied record gives no isoform-specific IHC pattern or epitope map, so it cannot predict which isoform an antibody detects (UniProt P51159; supplied record). |
| Where should IF/ICC signal appear? | HPA's ICC-IF entry reports staining in the mid piece and principal piece of sperm (HPA: subcellular ICC-IF). That observation belongs to the separate IF/ICC context; use the tissue IHC profile for paraffin-section interpretation (HPA: tissue IHC; HPA: subcellular ICC-IF). The supplied record gives no basis for an IF/ICC protocol here. |
| Situation | Likely cause | Next action |
|---|---|---|
| The known-positive glandular cells are blank. | The expected High signal in specified glandular cells is absent (HPA: tissue IHC); a failed staining step or compromised section is possible (general IHC practice). | Check that the reference cells are present, review the same-run control, then inspect retrieval, primary-antibody use and chromogenic detection records (general IHC practice). Do not infer RAB27A-specific fixation sensitivity from the supplied evidence. |
| The section is broadly brown with little cellular contrast. | Diffuse background can arise from incomplete blocking or washing, or excessive reagent exposure (general IHC practice). It does not reproduce the cell-associated cytoplasmic profile (HPA: tissue IHC). | Examine a no-primary control and review blocking, reagent exposure and washes; rescore only where cell boundaries and cytoplasm remain interpretable (general IHC practice). |
| Signal is strongest in a population HPA calls Not detected. | Strong adipocyte or bronchial respiratory-epithelial staining conflicts with those HPA cell-specific calls (HPA: tissue IHC). Cross-reactivity or endogenous detection activity is possible (general IHC practice). | Confirm the cell identity and compare no-primary and detection controls. Reassess the antibody with an appropriate positive region before assigning the unexpected signal to RAB27A (general IHC practice). |
| Staining looks mainly nuclear or like a continuous cell-surface rim. | That distribution differs from HPA's cytoplasmic profile and UniProt's intracellular vesicle locations (HPA: tissue IHC; UniProt P51159). | Check focus, counterstain and compartment assignment, then compare control sections. Report the distribution as atypical if it persists rather than scoring it as the expected pattern (general IHC practice). |
| Two samples have different overall staining despite similar target-cell appearance. | HPA scores specific cell populations, and RAB27A staining varies among them: High in specified glandular cells, Medium in bone-marrow hematopoietic cells and Not detected in some others (HPA: tissue IHC). | Compare the same identified cell population and compartment across samples. Record intensity and the fraction of positive target cells separately, with the run control reviewed alongside them (general IHC scoring practice). |
Experiment evidence
IHC & IF images showing RAB27A staining
A01608-1 · IHCIHC analysis of RAB27A using anti-RAB27A antibody (A01608-1). RAB27A was detected in paraffin-embedded section of human lung cancer tissue.
A01608-1 · IHCIHC analysis of RAB27A using anti-RAB27A antibody (A01608-1). RAB27A was detected in paraffin-embedded section of human mammary cancer tissu
M01608 · IHCIHC analysis of RAB27A using anti-RAB27A antibody (M01608). RAB27A was detected in paraffin-embedded section of human tonsil tissues. Heat m
M01608 · IHCIHC analysis of RAB27A using anti-RAB27A antibody (M01608). RAB27A was detected in paraffin-embedded section of human tonsil tissues. Heat m
M01608 · IFIF analysis of RAB27A using anti-RAB27A antibody (M01608). RAB27A was detected in immunocytochemical section of MCF7 cells. Enzyme antigen r
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000069974-RAB27A/tissue/cerebral+cortex#imid_10507765
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000069974-RAB27A/tissue/colon#imid_10403441
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000069974-RAB27A/tissue/liver#imid_10403042
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000069974-RAB27A/tissue/kidney#imid_10406605
HPA · ICC-IF SpermHuman Protein Atlas immunofluorescence, Sperm, antibody HPA001333; green = RAB27A, blue = nucleus, red = microtubules.
HPA · ICC-IF SpermHuman Protein Atlas immunofluorescence, Sperm, antibody HPA001333; green = RAB27A, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RAB27A IHC & IF
🧪Run colon first; its glandular cells should stain (HPA: High in colon glandular cells). Use cervix glandular cells as a tissue negative (HPA: Not detected in cervix glandular cells); the supplied rows do not identify internal negative cells in colon, so assign them only after independent validation and expect no specific DAB signal in validated negative cells (standard IHC practice).
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RAB27A in Sperm, with annotated localisation: Mid piece (approved), Principal piece (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a concentration-matched rabbit IgG isotype control, and RAB27A knockout tissue or cells as a biological negative (caption: rabbit primary antibody; standard IHC practice). Check endogenous peroxidase and biotin background in colon because the reported detection uses a biotinylated secondary, streptavidin–biotin complex, and DAB (caption: detection method; standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected A01608-1 paraffin-section caption does not state a fixative (caption: fixative unreported). The reported IHC procedure uses citrate retrieval at pH 6 for 20 minutes, but the evidence does not establish whether retrieval is essential (caption: retrieval conditions). The supplied evidence does not establish that frozen sections or IF are easier; assess colon background from endogenous peroxidase and biotin with the specified chromogenic system (caption: detection method; standard IHC practice).
HPA tissue IHC evidence for RAB27A
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Prostate | Glandular cells | High | Protein (IHC) | HPA → |
| Rectum | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Endometrium | Cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RAB27A IHC Tips
Troubleshoot RAB27A staining in paraffin sections by checking retrieval, cell type, intracellular pattern, and controls before interpreting chromogenic signal.
How should I adjust retrieval when RAB27A staining is weak?
Start with heat-mediated retrieval in citrate buffer at pH 6 for 20 minutes (datasheet A01608-1). The selected paraffin-section example used this retrieval before overnight incubation at 4°C with 1 μg/ml antibody (datasheet A01608-1); match those conditions initially so changes can be interpreted. If staining remains weak, vary heating or cooling within a controlled series, and assess tissue preservation alongside signal (standard IHC practice). Keep a known positive section in every run and compare cellular staining with background, because stronger chromogen alone does not establish specific detection (standard IHC practice).
Could fixation explain weak or uneven RAB27A staining?
The selected tissue caption identifies a paraffin-embedded section but does not state its fixative, so target-specific fixation sensitivity is unknown (datasheet A01608-1). Record the actual fixative and fixation duration for each specimen, then compare sections processed with the same retrieval and detection settings (standard IHC practice). If a specimen stains unevenly, inspect morphology and compare central tissue with exposed edges before changing antibody concentration (standard IHC practice). Use a concurrently processed positive control to distinguish a specimen-wide processing problem from a change in the sampled cell population; do not infer fixation sensitivity from tissue-expression patterns (standard IHC practice).
What staining pattern should I expect in a positive RAB27A section?
Expect a cytoplasmic pattern in tissue IHC, with possible granular or vesicle-associated emphasis, rather than an exclusively nuclear signal (HPA: cytoplasmic expression in most tissues; UniProt P51159 subcellular localisation). RAB27A associates with melanosomes, late endosomes, lysosomes, and endosomal exocytic vesicles, and has no transmembrane segment (UniProt P51159 subcellular localisation and topology). Assess the pattern within identifiable cells, because adjacent immune cells may also contribute signal (HPA: cytoplasmic expression including immune cells). If staining forms a continuous cell-border rim, compare it with the expected intracellular pattern and the negative control before calling it positive (standard IHC practice; UniProt P51159 subcellular localisation).
Can an unknown epitope limit interpretation across RAB27A isoforms?
RAB27A has Long and Short isoforms, but the supplied antibody caption does not identify its epitope or establish equal recognition of both (UniProt P51159 isoforms; datasheet A01608-1). Its annotated modifications include N-terminal acetylation or phosphorylation and a C-terminal cysteine methyl ester; their effect on this antibody’s tissue staining is unknown (UniProt P51159 modified residues; datasheet A01608-1). When comparing specimens, hold retrieval at citrate pH 6 and detection conditions constant before attributing different intensities to isoform expression (datasheet A01608-1; standard IHC practice). Resolve an isoform-specific claim only with antibody epitope information or independent isoform-aware evidence (standard IHC practice).
How can IF help investigate an ambiguous chromogenic RAB27A pattern?
Use IF/ICC as a separate validation experiment, since the supplied paraffin-section caption documents chromogenic detection rather than an IF protocol (datasheet A01608-1). Multiplex RAB27A with a marker for the cell population under study, then assess signal inside the marked cells instead of relying on overlapping tissue regions (standard IF practice; HPA: cytoplasmic expression including immune cells). Select a fluorophore channel after checking tissue autofluorescence and include single-label controls when measuring overlap (standard IF practice). If the recognized epitope faces the cytosol, optimize permeabilisation for access; the supplied evidence does not define this antibody’s epitope orientation (standard IF practice; datasheet A01608-1).
What should I check when DAB background obscures RAB27A?
Compare the stained section with a no-primary control and inspect whether diffuse color follows tissue edges, damaged areas, or the expected cells (standard IHC practice). The selected example used a biotinylated secondary, a streptavidin–biotin detection complex, and DAB, so assess detection-reagent background alongside primary-antibody background (datasheet A01608-1). Include an appropriate endogenous peroxidase block and verify that the block does not obscure tissue morphology; these are general chromogenic-IHC steps, not RAB27A-specific findings (standard IHC practice). If background persists, adjust blocking, washes, or antibody concentration one variable at a time while retaining a positive section (standard IHC practice).
How should I quantify RAB27A staining across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because RAB27A tissue staining is reported as cytoplasmic and may include immune cells (HPA: tissue IHC profile). For an intensity-based comparison, record an H-score from 0–300, using the percentage of cells at each intensity; alternatively report the percentage of positive cells or positive-cell density per mm² (standard IHC practice). Normalize counts to the area and number of eligible cells in the same annotated region, and keep thresholds and imaging settings fixed across specimens (standard IHC practice). Report epithelial and immune-cell results separately when both are present (standard IHC practice; HPA: cytoplasmic expression including immune cells).
When is apparent RAB27A positivity more likely to be artefactual?
Treat reproducible intracellular staining in an expected cell population as more persuasive than isolated nuclear staining, since RAB27A is associated with intracellular vesicle compartments and HPA reports cytoplasmic tissue expression (UniProt P51159 subcellular localisation; HPA: tissue IHC profile). Check which cells carry signal: HPA reports high staining in several gastrointestinal glandular-cell populations and medium staining in bone-marrow hematopoietic cells, while selected cell populations are not detected (HPA: tissue IHC levels). Discount staining confined to tissue edges or necrotic regions unless it persists in well-preserved areas (standard IHC practice). Compare no-primary and peroxidase-block controls before interpreting DAB deposits as antigen signal (standard IHC practice).
Boster reagents
Best RAB27A / Ras-related protein Rab-27A IHC Antibodies
The catalog antibodies show paraffin-section IHC in human, mouse, and rat samples, plus IF/ICC in human MCF7 cells (catalog: A01608-1 and M01608 IHC captions; M01608 IF caption).
Real IHC data
Anti-RAB27A Antibody ®
Cat # A01608-1
Real IHC data
Anti-RAB27A Antibody ® (monoclonal, 2F5)
Cat # M01608
A01608-1 will render with IHC images from human lung and mammary cancer and rat small intestine and spleen paraffin sections (A01608-1 IHC captions). M01608 will render with IHC images from human tonsil and prostatic cancer and mouse intestine paraffin sections; its separate IF image shows MCF7 cells (M01608 IHC and IF captions).
Which to pick: For tissue IHC, choose the rabbit antibody A01608-1 for the documented human and rat paraffin sections, or the mouse monoclonal M01608 for the documented human and mouse paraffin sections (catalog: hosts; A01608-1 and M01608 IHC captions). For IF/ICC, choose M01608 because its IF image documents MCF7 cells; A01608-1 lists ICC but has no IF image (catalog: applications and IF captions). Both list human, mouse, and rat reactivity, while their pictured cross-species IHC examples differ; neither IHC caption reports the fixative (catalog: reactivity; A01608-1 and M01608 IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P51159 (RB27A_HUMAN, Ras-related protein Rab-27A).
- Human Protein Atlas. RAB27A tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. RAB27A subcellular location (ICC-IF): Localized to the mid piece and principal piece..
- Human Protein Atlas. RAB27A antibody validation summary (2 antibodies).
- Rab27a was identified as a prognostic biomaker by mRNA profiling, correlated with malignant progression and subtype preference in gliomas. PloS one 2014 — PMC3935941.
- Overexpression of RAB27A in Oral Squamous Cell Carcinoma Promotes Tumor Migration and Invasion via Modulation of EGFR Membrane Stability. International journal of molecular sciences 2023 — PMC10488256.
- SH3BGRL3 promotes radioresistance and immune evasion in triple-negative breast cancer by regulating Rab27a. Biology direct 2026 — PMC13251207.
- KCa3.1 Promotes Proinflammatory Exosome Secretion by Activating AKT/Rab27a in Atrial Myocytes during Rapid Pacing. Cardiovascular therapeutics 2023 — PMC10079387.
- PubMed PMID:7592656 — UniProt-cited evidence.
- PubMed PMID:9066979 — UniProt-cited evidence.
- PubMed PMID:10571040 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RAB27A (Ras-related protein Rab-27A). Expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet A01608-1). Validated IHC antibody: A01608-1 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular 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 A01608-1); 2. Fixation: Image fixative and duration unreported (datasheet A01608-1); 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: Citrate pH 6 , 20 min (datasheet A01608-1); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A01608-1); 8. Primary antibody: Rabbit anti-RAB27A, 0.5-1μg/ml (datasheet A01608-1); 9. Primary incubation: Overnight at 4 °C (datasheet A01608-1). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A01608-1); Caveat: Glandular staining varies; cervix is undetected (HPA tissue IHC); Isoform / epitope: Long and Short isoforms; no transmembrane segment; epitope coverage unknown (UniProt); Regulation: Low expression in kidney and muscle (UniProt). Evidence: UniProt P51159, HPA tissue IHC · Enhanced, HPA ICC-IF · mid piece and principal piece., Datasheet A01608-1, PMC3935941, PMC13251207. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RAB27A/immunohistochemistry-ihc-guide.