This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
ROCK2 / Rho-associated protein kinase 2 · IHC design guide
Design Immunohistochemistry for ROCK2
Plan ROCK2 chromogenic IHC on paraffin sections with the catalog antibody at 0.5–1 μg/mL (datasheet PB9387). Assess cytoplasmic staining against tissue controls, bearing in mind the low consistency between antibody staining and RNA expression (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ROCK2 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 across most tissues (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet PB9387) | |
| Positive control | Testis+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 paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Antibody staining and RNA show low consistency (HPA tissue IHC) | |
| Regulation | No expression regulator specified (UniProt) | |
| Isoform / epitope | No isoforms or processing annotated; epitope unmapped (UniProt) |
Section 1
Recommended ROCK2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: PB9387) is accompanied by 3 published ROCK2 IHC protocols (PMC5513090; PMC6069530; PMC6372474).
| Sample | Paraffin-embedded Human Mammary Cancer tissue; fixative not specified (datasheet PB9387) |
| Fixation | Image fixative and duration unreported (datasheet PB9387); 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 PB9387); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9387) |
| Primary antibody | Rabbit anti-ROCK2, 0.5-1μg/ml (datasheet PB9387) |
| Primary incubation | Overnight at 4 °C (datasheet PB9387) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9387) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ROCK2-positive staining in leydig cells of testis (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (datasheet: PB9387); the vascular tumor study used a different retrieval reagent (PMC5513090).
Section 2
What Is the Expected ROCK2 Staining Pattern?
ROCK2 staining should be predominantly cytoplasmic across most tissues, with high staining in testicular Leydig cells and medium staining in several epithelial and neuronal cell populations (HPA tissue IHC). ROCK2 has no transmembrane segment, although UniProt also lists membrane, nuclear, and cytoskeletal associations (UniProt O75116 topology and subcellular location). Interpret tissue patterns cautiously: HPA rates its IHC profile Approved but reports low consistency with RNA expression (HPA tissue IHC).
| Leydig cells show strong cytoplasmic chromogen, with surrounding structures distinguishable. | This matches the clearest supplied positive reference: high Leydig cell staining in testis (HPA tissue IHC). Compare intensity within identified cells rather than calling an entire section uniformly positive (standard IHC practice). |
| Neuronal cells in cerebral cortex or glandular cells in appendix show moderate cytoplasmic staining. | These are medium examples, consistent with HPA’s broad cytoplasmic profile (HPA tissue IHC). A weaker result than the Leydig cell reference can therefore be expected without implying assay failure (HPA tissue IHC). |
| Signal is confined to nuclei or outlines every cell membrane, with little cytoplasmic staining. | Question the pattern against HPA’s cytoplasmic tissue profile (HPA tissue IHC). UniProt lists nuclear and membrane associations, so those compartments alone do not prove artefact; check whether the cell distribution and controls support the result (UniProt O75116 subcellular location; standard IHC practice). |
| Prominent staining appears in adipocytes or cardiomyocytes, which are listed as undetected. | Consider cross-reactivity or endogenous detection activity, and examine negative controls (HPA tissue IHC; standard IHC practice). HPA’s not detected calls describe those specific cells; they do not make the entire adipose or heart section a universal negative control (HPA tissue IHC). |
| Weak chromogen covers tissue and empty areas, or a known-positive Leydig cell section has no signal. | Diffuse background weakens confidence in cellular localisation; absence of the high reference calls for a control and workflow review (standard IHC practice; HPA tissue IHC). Neither observation alone establishes ROCK2 absence or a target-specific fixation effect (standard IHC practice). |
💡Expected ROCK2 appearanceCall a result positive when identified cells show predominantly cytoplasmic staining—strong in Leydig cells or moderate in listed positive populations—while diffuse background or indiscriminate staining of listed undetected cells raises concern (HPA tissue IHC; standard IHC practice).
| Tissue and cell choice | Use Leydig cells as a high reference and, if useful, appendix glandular or cerebral cortex neuronal cells as medium comparators (HPA tissue IHC). Score the named cell population, since the listed levels do not describe every cell in a tissue (HPA tissue IHC). |
| Compartment and protein topology | Expect a mainly cytoplasmic IHC pattern (HPA tissue IHC). ROCK2 lacks a transmembrane segment, yet UniProt records plasma membrane, actin-associated, and nuclear localisation; isolated membrane or nuclear signal needs contextual review rather than automatic acceptance or rejection (UniProt O75116 topology and subcellular location). |
| Antibody validation and tissue concordance | HPA lists HPA007459 and CAB008666 as IHC Approved, while its tissue profile notes low staining-to-RNA consistency (HPA antibodies; HPA tissue IHC). Approval supports use as a reference point, but discordant cellular patterns still warrant controls (HPA tissue IHC; standard IHC practice). |
| Processing and epitope interpretation | UniProt lists one 1–1388 chain, no signal peptide or propeptide, and no glycosylation sites (UniProt O75116 processing and glycosylation). The payload gives no epitope position or target-specific fixation sensitivity, so it cannot predict a retrieval setting or processing-dependent pattern. |
| IF/ICC cross-check | Q: What should IF/ICC show? A: Predominantly cytosolic ROCK2; HPA marks that location Supported and lists HPA044109 as ICC Supported (HPA subcellular; HPA antibodies). Use this as a localisation comparison, not an IHC intensity standard (HPA subcellular; HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| No staining in Leydig cells of a testis positive control. | The expected high reference is missing (HPA tissue IHC); the slide alone cannot identify the failed step (standard IHC practice). | Check that the intended cells are present, then review primary antibody use, retrieval, detection reagents, and control slide performance (standard IHC practice). Do not infer ROCK2 absence from this run. |
| All compartments show a diffuse brown haze. | Non-specific antibody or detection background can obscure a cellular pattern (standard IHC practice). | Inspect the no-primary control; review blocking, washes, primary antibody concentration, and chromogen development against the kit instructions (standard IHC practice). Reassess cytoplasmic localisation after background is controlled (HPA tissue IHC). |
| Brown deposits persist in a no-primary control. | Endogenous detection activity or a detection-reagent problem may contribute signal independently of the primary antibody (standard IHC practice). | Check the detection chemistry and its matching endogenous-activity block, then repeat the control alongside the test slide (standard IHC practice). |
| Adipocytes or cardiomyocytes stain strongly despite the expected negative reference. | HPA lists these cells as not detected; cross-reactivity or endogenous detection activity is possible, though HPA reports low tissue-profile concordance (HPA tissue IHC; standard IHC practice). | Confirm cell identity and compare a no-primary control and a listed positive cell population in the same run (standard IHC practice; HPA tissue IHC). Treat disagreement as unresolved until controls clarify it. |
| Only nuclei or continuous membrane rims stain across many cell types. | That distribution departs from HPA’s predominantly cytoplasmic tissue pattern, although UniProt permits nuclear and membrane association (HPA tissue IHC; UniProt O75116 subcellular location). | Review counterstain and morphology, compare the listed positive cells, and check the no-primary control before scoring compartment-specific staining (standard IHC practice; HPA tissue IHC). |
| A medium-reference tissue looks weaker than the Leydig cell control. | Different reported levels are expected: Leydig cells are high, while appendix glandular and cerebral cortex neuronal cells are medium (HPA tissue IHC). | Score intensity in the specified cell types with the same run’s controls (standard IHC practice). Investigate only if the positive control fails or the expected cellular pattern is lost (HPA tissue IHC; standard IHC practice). |
Experiment evidence
IHC & IF images showing ROCK2 staining
PB9387 · IHCIHC analysis of ROCK2 using anti-ROCK2 antibody (PB9387). ROCK2 was detected in a paraffin-embedded section of Human Mammary Cancer tissue.
PB9387 · IHCIHC analysis of ROCK2 using anti-ROCK2 antibody (PB9387). ROCK2 was detected in a paraffin-embedded section of Mouse Cardiac Muscle tissue.
A01023-1 · IFIF analysis of ROCK2 using anti-ROCK2 antibody (A01023-1). ROCK2 was detected in immunocytochemical section of Hep cells. Enzyme antigen ret
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000134318-ROCK2/tissue/cerebral+cortex#imid_2477968
HPA · StomachHuman Protein Atlas IHC, Stomach. Source: https://www.proteinatlas.org/ENSG00000134318-ROCK2/tissue/stomach+1#imid_2477852
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000134318-ROCK2/tissue/colon#imid_2477878
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000134318-ROCK2/tissue/liver#imid_2477887
HPA · ICC-IF HeLaHuman Protein Atlas immunofluorescence, HeLa, antibody HPA044109; green = ROCK2, blue = nucleus, red = microtubules.
HPA · ICC-IF HeLaHuman Protein Atlas immunofluorescence, HeLa, antibody HPA044109; green = ROCK2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ROCK2 IHC & IF
🧪Run testis first; Leydig cells should stain strongly (HPA: High in Leydig cells). Use adipose tissue as the negative, with adipocytes expected to lack detectable staining (HPA: Not detected in adipocytes); on the testis slide, assess cells outside the Leydig-cell compartment for background, without assuming they are ROCK2-negative (HPA: Leydig-cell staining reported).
Positive control tissue: Testis (Leydig cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ROCK2 in HeLa, Hep-G2, U2OS, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), NIH 3T3, with annotated localisation: Cytosol (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a host- and clonality-matched rabbit IgG isotype control, and ROCK2-knockout tissue if available; a peptide block is an alternative when the immunizing peptide is available (caption: rabbit anti-ROCK2; standard IHC practice). For the Leydig-cell-positive testis slide, quench endogenous peroxidase and check for endogenous biotin background before interpreting DAB signal from the biotin-based detection system (HPA: High in Leydig cells; caption: SABC with DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected paraffin-section caption does not state its fixative (caption: fixative unreported). The documented IHC workflow used heat retrieval in EDTA at pH 8.0, so use that as the starting retrieval condition; frozen sections and IF/ICC are not established as easier by the supplied evidence (caption: EDTA retrieval; HPA: cytosolic ICC-IF localization). In testis, distinguish Leydig-cell DAB staining from pigment and background by comparison with the no-primary slide (HPA: High in Leydig cells; standard IHC practice).
HPA tissue IHC evidence for ROCK2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low 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 |
|---|---|---|---|---|
| Testis | Leydig cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Kidney | Cells in glomeruli | Not detected | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Oral mucosa | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ROCK2 IHC Tips
Troubleshoot ROCK2 staining in paraffin sections by checking retrieval, compartment, controls and cell-specific scoring before interpreting chromogenic signal.
What retrieval conditions should I start with for ROCK2 paraffin sections?
Use heat-mediated antigen retrieval in EDTA buffer at pH 8.0 for paraffin sections (datasheet PB9387). The documented ROCK2 tissue image used that retrieval before incubation with 1 μg/ml antibody overnight at 4°C (datasheet PB9387). If staining is weak, compare retrieval duration on serial sections while holding antibody concentration, detection and development constant (standard IHC practice). Include a positive control section and a section processed without primary antibody so that improved signal can be distinguished from increased background (standard IHC practice). The caption does not specify a retrieval heating time (datasheet PB9387).
How should I assess whether fixation is limiting ROCK2 staining?
ROCK2-specific sensitivity to fixation is unknown from the supplied evidence, and the selected paraffin-section caption does not state a fixative (datasheet PB9387). Record the specimen fixative and fixation duration, then compare similarly processed positive-control sections across runs (standard IHC practice). If a section stains weakly, evaluate retrieval and antibody titration alongside fixation history before assigning the cause (standard IHC practice). Avoid treating a difference between specimens as a ROCK2-specific fixation effect without a controlled comparison (standard IHC practice). The documented pH 8.0 EDTA retrieval provides a starting condition for that comparison (datasheet PB9387).
Which compartments should show credible ROCK2 staining?
Expect predominantly cytoplasmic staining in tissue sections (HPA: cytoplasmic expression in most tissues), consistent with a supported cytosolic location in cell imaging (HPA: Cytosol supported). ROCK2 is also annotated at the cell membrane, nucleus and centrosome, with associations to actin microfilaments and the plasma membrane (UniProt O75116: subcellular location). Assess whether staining follows recognizable cell boundaries or cytoplasm under the same counterstain and exposure conditions (standard IHC practice). A strong isolated nuclear or surface-only pattern needs independent validation before being assigned to ROCK2, because those locations are annotations rather than the predominant tissue-IHC pattern (UniProt O75116: subcellular location; HPA: tissue profile).
Could epitope choice explain discordant ROCK2 staining?
The supplied ROCK2 record lists 0 isoforms and a single annotated chain spanning residues 1–1388 (UniProt O75116: isoforms and processing). Its kinase domain spans 92–354, Rho-binding domain 979–1047 and PH domain 1150–1349 (UniProt O75116: domains). The record also notes phosphorylation at residues including 414, 722 and 1137 (UniProt O75116: modified residues). Check the antibody's disclosed immunogen or epitope before interpreting discordance between antibodies, and compare staining on adjacent sections under matched retrieval and detection conditions (standard IHC practice). Do not assume that the catalog antibody detects a particular phospho-state without epitope-specific validation (standard IHC practice).
How can I check a ROCK2 pattern by immunofluorescence?
Use IF as a separate validation experiment and multiplex ROCK2 with a marker identifying the cell population under study (standard IF practice). Choose a fluorophore channel after examining unstained tissue autofluorescence, and include single-label controls to assess bleed-through (standard IF practice). Because ROCK2 lacks a transmembrane segment and has a supported cytosolic location, use permeabilisation when testing an intracellular ROCK2 epitope (UniProt O75116: topology; HPA: Cytosol supported; standard IF practice). Verify the antibody's documented IF suitability and epitope accessibility before transferring an IHC staining condition to IF (standard IF practice). Compare the resulting cellular pattern with the predominantly cytoplasmic tissue-IHC profile (HPA: tissue profile).
What should I change when ROCK2 DAB staining is diffuse?
The documented ROCK2 paraffin-section image used 10% goat serum blocking, a biotinylated secondary antibody and DAB development (datasheet PB9387). For diffuse brown signal, inspect a no-primary control and shorten DAB development or titrate the primary antibody while preserving a positive control (standard IHC practice). Apply a peroxidase block before chromogenic detection and consider endogenous biotin when using a biotin-based system (standard IHC practice). Compare background in acellular areas, section edges and expected low-signal cells with staining in intact cells (standard IHC practice). HPA reports adipocytes and cardiomyocytes as not detected, though those observations alone do not validate a local negative control (HPA: tissue IHC).
How should I quantify ROCK2 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, then record the percentage of positive cells and staining intensity for each specimen (standard IHC practice). An H-score combines intensity grades 0–3 with their cell percentages and ranges from 0–300 (standard IHC practice). Normalise positive-cell counts to the number of eligible cells, or report positive-cell density per mm² of viable tissue when cell counting is impractical (standard IHC practice). Keep retrieval, chromogen development, imaging and scoring thresholds consistent across the comparison (standard IHC practice). Report cell type because HPA lists high staining in Leydig cells but no detected staining in several other cell populations (HPA: tissue IHC).
How do I distinguish ROCK2 signal from a staining artefact?
A credible ROCK2 result should show cellular staining with a chiefly cytoplasmic pattern and appropriate control behavior (HPA: tissue profile; standard IHC practice). Check whether signal occurs in plausible cells: HPA reports high staining in Leydig cells and medium staining in breast glandular cells, but no detected staining in adipocytes (HPA: tissue IHC). Treat edge-restricted color, necrotic deposits and staining reproduced in a no-primary control as possible processing or detection artefacts (standard IHC practice). Exclude endogenous peroxidase activity before interpreting DAB signal, and verify unusual nuclear-only patterns with an independent control (standard IHC practice; UniProt O75116: subcellular location). HPA rates its tissue staining Approved but notes low consistency with RNA expression (HPA: reliability).
Boster reagents
Best ROCK2 / Rho-associated protein kinase 2 IHC Antibodies
Anti-ROCK2 antibodies have IHC images from human mammary cancer, mouse cardiac muscle, and rat brain paraffin sections (PB9387 image captions), and an IF/ICC image from Hep cells (A01023-1 image caption).
Real IHC data
Anti-ROCK2 Antibody ®
Cat # PB9387
Real IF data
Anti-ROCK2 Antibody ®
Cat # A01023-1
PB9387 is listed for IHC and has paraffin-section images from human mammary cancer, mouse cardiac muscle, and rat brain (PB9387 applications and image captions). A01023-1 is listed for IF/ICC and has an image from Hep cells (A01023-1 applications and image caption).
Which to pick: Choose PB9387 for tissue IHC: its paraffin-section images use heat retrieval in EDTA at pH 8.0 and 1 μg/ml primary antibody, while the fixative is unreported (PB9387 image captions). Choose A01023-1 for IF/ICC: its Hep-cell image uses 5 μg/ml primary antibody; its fixative is unreported (A01023-1 image caption). For IHC across species, PB9387 has image evidence in human, mouse, and rat tissue; A01023-1 lists the same species as reactive, but its supplied image demonstrates IF/ICC in Hep cells (PB9387 image captions; A01023-1 reactivity and image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O75116 (ROCK2_HUMAN, Rho-associated protein kinase 2).
- Human Protein Atlas. ROCK2 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ROCK2 subcellular location (ICC-IF): Localized to the cytosol..
- Human Protein Atlas. ROCK2 antibody validation summary (3 antibodies).
- Rho kinase proteins display aberrant upregulation in vascular tumors and contribute to vascular tumor growth. BMC cancer 2017 — PMC5513090.
- ROCK signaling promotes collagen remodeling to facilitate invasive pancreatic ductal adenocarcinoma tumor cell growth. EMBO molecular medicine 2017 — PMC5286371.
- Overexpression of Rho-Associated Coiled-Coil Containing Protein Kinase 2 Is Correlated with Clinical Progression and Poor Prognosis in Breast Cancer. Medical science monitor : international medical journal of experimental and clinical research 2018 — PMC6069530.
- The interaction of Lin28A/Rho associated coiled-coil containing protein kinase2 accelerates the malignancy of ovarian cancer. Oncogene 2019 — PMC6372474.
- PubMed PMID:9933571 — UniProt-cited evidence.
- PubMed PMID:9734811 — UniProt-cited evidence.
- PubMed PMID:15815621 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ROCK2 (Rho-associated protein kinase 2). Expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PB9387). Validated IHC antibody: PB9387 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Leydig cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded Human Mammary Cancer tissue; fixative not specified (datasheet PB9387); 2. Fixation: Image fixative and duration unreported (datasheet PB9387); 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 PB9387); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9387); 8. Primary antibody: Rabbit anti-ROCK2, 0.5-1μg/ml (datasheet PB9387); 9. Primary incubation: Overnight at 4 °C (datasheet PB9387). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Antibody staining and RNA show low consistency (HPA tissue IHC); Isoform / epitope: No isoforms or processing annotated; epitope unmapped (UniProt); Regulation: No expression regulator specified (UniProt). Evidence: UniProt O75116, HPA tissue IHC · Approved, HPA ICC-IF · cytosol., Datasheet PB9387, PMC5513090, PMC6069530. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ROCK2/immunohistochemistry-ihc-guide.