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RAB27A / Ras-related protein Rab-27A · IHC design guide
Design Immunohistochemistry for RAB27A
Plan RAB27A paraffin IHC using glandular cell positive controls and cell populations reported as undetected (HPA tissue IHC). The guide covers fixation consistency, cytoplasmic scoring, and interpretation of variable tissue staining (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 staining in glandular and 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 | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA expression show medium consistency (HPA tissue IHC) | |
| Regulation | Expression varies by tissue (HPA tissue IHC) | |
| Isoform / epitope | Long and Short isoforms; epitope impact unknown (UniProt) |
Section 1
Recommended RAB27A IHC & IF Protocols
The catalog antibody uses citrate pH 6 retrieval (datasheet A01608-1). Published RAB27A IHC protocols cover glioma, breast tumors, and canine atrium (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 noteStart with citrate pH 6 retrieval for the catalog antibody (datasheet A01608-1). EDTA pH 8.0 was reported for glioma sections with a different antibody (PMC3935941).
Section 2
What Is the Expected RAB27A Staining Pattern?
RAB27A should appear mainly as cytoplasmic staining in tissue IHC, often associated with intracellular vesicles; UniProt places it at melanosomes, late endosomes, lysosomes and endosomal exocytic vesicles, and reports no transmembrane segment (HPA: tissue IHC profile; UniProt P51159: location and topology). Expect strong staining in glandular cells of several digestive tissues and detectable staining in some immune cells. HPA rates its tissue IHC reliability Enhanced, with medium consistency against RNA data (HPA: tissue IHC).
| Glandular cells show cytoplasmic chromogen in stomach, colon or small intestine. | This fits the reported High glandular-cell staining in those tissues (HPA: tissue IHC). Record the fraction and intensity of positive cells; do not require every cell in a section to stain identically (general IHC practice). |
| A section shows predominantly nuclear staining with little cytoplasmic signal. | That does not match the reported cytoplasmic tissue profile or intracellular membrane locations (HPA: tissue IHC; UniProt P51159: location). Treat it as suspect until controls and staining conditions are checked (general IHC practice). |
| Adipocytes or bronchial respiratory epithelial cells stain as strongly as the intended positive cells. | Those specific cell populations are listed as Not detected (HPA: tissue IHC). Consider antibody cross-reactivity or endogenous detection activity, and compare with an appropriate negative control (general IHC practice). |
| Chromogen coats many structures without clear cell boundaries or compartment contrast. | Diffuse background cannot establish RAB27A localisation (general IHC practice). Assess nonspecific binding, residual detection activity and the control slide before scoring individual cells (general IHC practice). |
| Glandular cells in a known-positive stomach or colon section have no visible signal. | Both are reported High in glandular cells (HPA: tissue IHC). First check that the expected cells are present, then review the IHC-validated antibody and detection run; a blank slide alone does not establish absent RAB27A (general IHC practice). |
💡Expected RAB27A appearanceCall a result positive when glandular or other supported cells show discernible cytoplasmic staining, with strong glandular-cell signal expected in the listed High tissues; isolated nuclear-only staining or staining equally strong in listed Not detected cell populations is suspect (HPA: tissue IHC; UniProt P51159: location).
| Cell population within a tissue | Interpret the named cell type, not the entire organ: kidney glomerular cells and bronchial respiratory epithelial cells are Not detected, while stomach glandular cells are High (HPA: tissue IHC). |
| Intracellular location | RAB27A is associated with melanosomes, late endosomes, lysosomes and exocytic vesicles, and has no transmembrane segment (UniProt P51159: location and topology). Cytoplasmic staining is the tissue-level expectation (HPA: tissue IHC); IHC alone need not resolve each vesicle (general IHC practice). |
| Tissue and immune-cell context | Several digestive glandular populations are High, bone-marrow hematopoietic cells are Medium, and the overall profile includes immune cells (HPA: tissue IHC). UniProt also reports expression in cytotoxic T lymphocytes and mast cells (UniProt P51159: tissue specificity). |
| Strength of the reference pattern | HPA calls tissue IHC reliability Enhanced but describes only medium consistency between staining and RNA expression (HPA: tissue IHC). Two listed antibodies have Enhanced IHC status, HPA001333 and CAB034046 (HPA: antibodies); use the reference pattern as evidence, not a guarantee for every specimen (general IHC practice). |
| Isoforms and specimen handling | UniProt lists Long and Short isoforms, but the supplied sources do not establish an isoform-specific IHC pattern or RAB27A-specific fixation sensitivity (UniProt P51159: isoforms; HPA: tissue IHC). Interpret a changed pattern cautiously rather than assigning it to an isoform or fixation effect. |
| IF/ICC Q: Is the tissue IHC pattern the expected IF pattern? | A: The supplied HPA ICC-IF record shows approved mid-piece and principal-piece localisation in sperm (HPA: subcellular ICC-IF). That observation does not define the appearance of paraffin tissue IHC; evaluate this page's chromogenic results against the tissue IHC profile (HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in glandular cells of a stomach or colon positive control. | A control expected to be High is blank (HPA: tissue IHC); the failed step cannot be identified from that observation alone (general IHC practice). | Confirm the glandular cells are present, then review the catalog antibody's IHC-P instructions, staining run and detection control before interpreting experimental negatives (general IHC practice). |
| All sections have a faint, widespread chromogen haze. | Widespread haze may reflect nonspecific binding or incomplete control of detection background (general IHC practice). | Review blocking, washes and the negative control; score RAB27A only where cellular staining remains distinguishable from background (general IHC practice). |
| A negative-control slide also develops chromogen. | Endogenous detection activity or reagent-related background may contribute signal independent of specific antibody binding (general chromogenic IHC practice). | Use the control result to assess endogenous activity and the detection reagents; resolve that signal before calling test-slide cells positive (general chromogenic IHC practice). |
| Nuclei stain more strongly than cytoplasm. | A nuclear-dominant pattern conflicts with the reported cytoplasmic IHC profile and intracellular organelle locations (HPA: tissue IHC; UniProt P51159: location). | Check counterstain and negative controls, then compare a known-positive tissue; do not score isolated nuclear colour as the expected RAB27A pattern (general IHC practice). |
| Adipocytes or bronchial respiratory epithelial cells appear strongly positive. | These specific cells are listed as Not detected (HPA: tissue IHC); cross-reactivity or endogenous detection activity is possible (general IHC practice). | Verify cell identity, compare a negative control and examine whether the supported positive cell population retains a distinct cytoplasmic pattern (HPA: tissue IHC; general IHC practice). |
| A whole kidney or brain section is labelled negative from one cell population. | HPA's kidney Not detected entry applies to glomerular cells, while its listed brain populations have Low staining; these entries do not classify every cell in either organ (HPA: tissue IHC). | Record the cell population and compartment actually examined, and compare that population with its corresponding HPA entry before assigning an expected level (HPA: tissue IHC; general IHC 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 the tissue negative (HPA: Not detected in cervix glandular cells); on the colon slide, any cells used as internal negatives should show no cell-associated DAB above the no-primary background, without assuming that all non-glandular cells are RAB27A-negative.
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- and clonality-matched rabbit IgG isotype control, and a validated RAB27A-knockout specimen as a biological negative (A01608-1 caption: rabbit primary). For colon IHC, quench endogenous peroxidase and check for endogenous biotin background with the biotinylated secondary and SABC/DAB detection used in the selected example (A01608-1 caption).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected paraffin-section caption does not state the fixative (A01608-1 caption: fixative unreported). The example used heat-mediated citrate retrieval at pH 6 for 20 minutes, but it does not establish that retrieval is required (A01608-1 caption). The supplied evidence does not show that frozen sections or IF/ICC are easier; for colon IHC, distinguish possible luminal or mucus-associated DAB deposits from glandular-cell staining (HPA: High in colon glandular cells; A01608-1 caption: DAB detection).
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 section chromogenic IHC, using the selected antibody’s tissue protocol and cell type specific expression as reference points.
What retrieval should I start with if RAB27A staining is weak?
Start with heat mediated retrieval in citrate buffer at pH 6 (datasheet A01608-1). The selected antibody’s paraffin section image used 20 minutes of retrieval before staining human lung cancer tissue (datasheet A01608-1). Run a known positive section alongside the test section and keep heating and cooling conditions consistent across both. If staining remains weak, compare a modestly longer retrieval time on matched sections while watching for tissue damage; this is an optimization step, not a demonstrated requirement for RAB27A. Recheck primary antibody concentration against the documented 1 μg/ml condition before attributing weak signal to retrieval (datasheet A01608-1).
Could fixation explain inconsistent RAB27A staining between paraffin sections?
Target specific fixation sensitivity is unknown: the selected paraffin section caption does not report a fixative (datasheet A01608-1). Record fixative, fixation duration, tissue thickness and processing history for each specimen, then compare sections processed together when investigating inconsistent staining. Keep retrieval at citrate pH 6 and the documented 20 minutes initially, so a change in staining can be interpreted against the selected antibody’s procedure (datasheet A01608-1). If archival blocks differ in processing, include a concurrently stained reference section and inspect morphology before changing antibody concentration. Treat any apparent fixation effect as an observation requiring controlled comparison, rather than an established property of RAB27A.
Where should convincing RAB27A signal appear in a tissue section?
Evaluate RAB27A primarily as cytoplasmic staining within identifiable cells, consistent with the tissue staining profile (HPA: cytoplasmic expression in most tissues). Its association with melanosomes, late endosomes, lysosomes and endosomal exocytic vesicles makes granular or uneven cytoplasmic signal plausible (UniProt P51159 subcellular location). Because RAB27A lacks a transmembrane segment, a sharply continuous plasma membrane outline should prompt review of antibody specificity and detection artefacts (UniProt P51159 topology). Compare the pattern with an expected positive cell population, such as glandular cells in stomach, and inspect a matched negative control (HPA: High in stomach glandular cells). Avoid assigning a vesicle identity from chromogenic staining alone.
How could isoforms or epitope accessibility affect RAB27A IHC?
RAB27A has Long and Short isoforms, so check whether the antibody’s stated immunogen covers both before interpreting a negative section as absence of the protein (UniProt P51159 isoforms). The supplied tissue caption establishes staining at 1 μg/ml after citrate pH 6 retrieval, but does not identify the recognized epitope (datasheet A01608-1). Document the immunogen sequence if available and avoid claiming isoform specificity without mapping or direct validation. RAB27A also has annotated modifications near residue 2 and at residue 221; their effect on this antibody’s binding is unknown (UniProt P51159 modified residues). Compare retrieval conditions and an independent antibody before attributing discordant staining to an isoform or modification.
How should I investigate RAB27A localisation with complementary IF?
Use IF as a complementary localisation experiment, while interpreting this page’s documented staining conditions as paraffin section chromogenic IHC conditions (datasheet A01608-1). For glandular tissue, multiplex RAB27A with an epithelial marker such as cytokeratin to identify the cell population associated with strong tissue staining (HPA: High in stomach glandular cells). Choose spectrally separated fluorophores after checking tissue autofluorescence in an unstained control, and include single stain controls when assigning colocalisation. Since RAB27A has no transmembrane segment and associates with intracellular compartments, optimize gentle permeabilisation to expose its intracellular epitope without washing away spatial detail (UniProt P51159 topology and subcellular location). Confirm any puncta with appropriate controls before assigning them to a particular organelle.
How can I distinguish RAB27A signal from chromogenic background?
First inspect a no primary control for endogenous enzyme activity, nonspecific secondary binding and pigment that could resemble DAB. The selected paraffin section procedure used 10% goat serum blocking, a biotinylated secondary and DAB detection, so background can arise at several detection steps (datasheet A01608-1). Apply a standard endogenous peroxidase block for chromogenic IHC, and assess a secondary only control if diffuse staining persists; these are workflow controls, not reported RAB27A requirements. Compare signal in high expressing glandular cells with adipocytes reported as not detected, while accounting for the different cell types (HPA: High in stomach glandular cells; HPA: Not detected in adipocytes). Reassess excessive primary concentration or development time if controls also stain.
What is a defensible way to quantify RAB27A IHC staining? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, because RAB27A staining is cytoplasmic across multiple tissue cell types (HPA: cytoplasmic expression in most tissues). For glandular cells, report the percentage of positive cells and an H-score from 0–300, calculated from the percentages at intensity grades 0–3. Normalize cell counts to the number of evaluable cells of that type; for dispersed positive cells, report density per mm² of viable tissue instead. Apply one exposure and scoring threshold to all sections, and exclude necrotic or damaged areas by a prespecified rule. Include reference sections across staining runs and report observer or image analysis agreement rather than treating raw DAB darkness as protein concentration.
When should a positive RAB27A stain be considered artefactual?
A credible result has intracellular cytoplasmic staining in a plausible cell population, consistent with the tissue profile and RAB27A’s endosomal and lysosomal associations (HPA: cytoplasmic expression in most tissues; UniProt P51159 subcellular location). Question isolated nuclear staining or a uniform cell surface rim, given the annotated intracellular locations and lack of a transmembrane segment (UniProt P51159 subcellular location and topology). Check whether staining concentrates at section edges, in necrotic regions or in areas positive on a no primary control; these patterns suggest processing or detection artefacts. Compare glandular cells reported as high with adipocytes reported as not detected, but interpret each compartment separately (HPA: High in stomach glandular cells; HPA: Not detected in adipocytes). Replicate the pattern before making biological claims.
Boster reagents
Best RAB27A / Ras-related protein Rab-27A IHC Antibodies
IHC images show RAB27A staining in human, mouse, and rat paraffin sections (catalog IHC captions: A01608-1, M01608); IF images show staining in MCF7 cells (catalog IF caption: M01608).
Real IHC data
Anti-RAB27A Antibody ®
Cat # A01608-1
Real IHC data
Anti-RAB27A Antibody ® (monoclonal, 2F5)
Cat # M01608
A01608-1 has IHC images from human lung and mammary cancers and rat small intestine and spleen (catalog IHC captions: A01608-1). M01608 has IHC images from human tonsil and prostatic cancer and mouse intestine, plus an IF image from MCF7 cells (catalog IHC/IF captions: M01608).
Which to pick: For paraffin-section IHC, choose rabbit-host A01608-1 or mouse monoclonal M01608 (catalog host/clone; catalog IHC captions: A01608-1, M01608). For IF/ICC, choose M01608: its applications include both, and its IF image shows MCF7 cells (catalog applications; catalog IF caption: M01608). For cross-species IHC, both list human, mouse, and rat reactivity, although the IHC images show human and rat tissue for A01608-1 and human and mouse tissue for M01608; neither product’s paraffin-section caption reports the fixative (catalog reactivity; catalog IHC captions: A01608-1, M01608).
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: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Staining and RNA expression show medium consistency (HPA tissue IHC); Isoform / epitope: Long and Short isoforms; epitope impact unknown (UniProt); Regulation: Expression varies by tissue (HPA tissue IHC). 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.