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ROCK1 / Rho-associated protein kinase 1 · IHC design guide
Design Immunohistochemistry for ROCK1
Plan ROCK1 IHC in paraffin sections around the cytoplasmic staining observed in most tissues (HPA tissue IHC). Compare cell types with high staining against those with undetected staining, and score the chromogenic signal by cell type (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ROCK1 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 | Predominantly cytoplasmic in tissue sections (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in most tissues (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet M00722) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Heart muscle+1 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); ROCK1-specific effects are unknown. | |
| Caveat | Staining varies by cell type within tissues (HPA tissue IHC) | |
| Regulation | No expression regulator established (UniProt) | |
| Isoform / epitope | No annotated isoforms or extracellular segment (UniProt) |
Section 1
Recommended ROCK1 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: M00722) is accompanied by published ROCK1 IHC conditions for prostate, corpora cavernosa, endometrial, and lymph-node tissue (PMC6781985; PMC12683496; PMC5647010; PMC5100726).
| Sample | Paraffin-embedded human kidney tissue; fixative not specified (datasheet M00722) |
| Fixation | Image fixative and duration unreported (datasheet M00722); 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 M00722) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M00722) |
| Primary antibody | Rabbit monoclonal (clone CDE-18) anti-ROCK1, 1:50 (datasheet M00722) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet M00722) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ROCK1-positive staining in lymphoid tissue of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated citrate retrieval at pH 6 (datasheet: M00722). For the prostate TMA protocol, use its reported Tris-EDTA-citrate retrieval at pH 7.8 (PMC6781985).
Section 2
What Is the Expected ROCK1 Staining Pattern?
ROCK1 is mainly cytoplasmic, with reported cytoskeletal, membrane, projection, centrosomal and minor Golgi associations; it has no transmembrane segment (UniProt Q13464). In IHC, expect cytoplasmic staining across many tissues, including strong staining in selected epithelial, hematopoietic, glomerular and macrophage populations (HPA tissue IHC). HPA rates its tissue staining “Approved,” with medium agreement with RNA data and external verification pending (HPA tissue IHC).
| Cytoplasmic staining is strong in bronchial respiratory epithelium, colonic glandular cells or lung macrophages. | This matches cell-specific high staining reported by HPA (HPA tissue IHC). Judge the named cells rather than the whole tissue: neighboring cells may differ. Confirm that the signal follows cell outlines and remains distinguishable from background (general IHC practice). |
| Signal is confined to nuclei, with little or no cytoplasmic staining in otherwise positive cells. | An exclusively nuclear pattern does not match HPA’s predominantly cytoplasmic tissue profile or UniProt’s listed locations (HPA tissue IHC; UniProt Q13464). Treat it as suspect and check localization with a second IHC-approved antibody and an appropriate positive section (HPA antibodies; general IHC practice). |
| Strong staining appears in cardiomyocytes or skeletal myocytes while suitable positive cells stain normally. | HPA reports ROCK1 as not detected in those cell types (HPA tissue IHC). Unexpected signal may reflect antibody cross-reactivity or endogenous detection activity; inspect adjacent cell types and detection controls before assigning it to myocytes (general IHC practice). |
| Brown signal coats many structures evenly or remains visible in areas without identifiable cells. | A uniform haze does not show the cell-specific cytoplasmic distribution described by HPA (HPA tissue IHC). Consider nonspecific binding, inadequate blocking or residual chromogen; compare a no-primary control and review washing and detection steps (general IHC practice). |
| No staining appears in a designated positive cell population, such as bone-marrow hematopoietic cells. | That conflicts with HPA’s high staining report for those cells (HPA tissue IHC). First confirm tissue identity and control performance, then review the antibody’s IHC-P instructions, retrieval, dilution and detection chemistry (general IHC practice). One negative section alone cannot establish absent ROCK1 expression. |
💡Expected ROCK1 appearanceCall a section positive when identifiable HPA high-expressing cells show distinct, predominantly cytoplasmic staining above background; isolated nuclear staining, uniform haze or strong signal in HPA-negative myocytes warrants investigation (HPA tissue IHC; UniProt Q13464; general IHC practice).
| Cell type within the tissue | HPA reports high signal in appendix lymphoid tissue, bone-marrow hematopoietic cells, bronchial epithelium, colonic and fallopian-tube glands, gallbladder glands, glomerular cells and lung macrophages (HPA tissue IHC). Assess the specified cells; a tissue-wide average can hide a valid focal pattern (general IHC practice). |
| Low and undetected comparators | Neuronal cells in hippocampus and caudate are low, while cardiomyocytes and skeletal myocytes are not detected in HPA tissue IHC (HPA tissue IHC). Use these as pattern comparisons, not proof that every cell on those sections must be unstained (general IHC practice). |
| Compartment and topology | ROCK1 is chiefly cytoplasmic; UniProt also lists cytoskeletal and cell-projection sites, centrosomal structures, cell membrane and a small Golgi-associated fraction (UniProt Q13464). It has no transmembrane segment (UniProt Q13464). Do not require a crisp membrane-only outline for a positive IHC call. |
| Strength of tissue-pattern evidence | Three listed antibodies have IHC “Approved” status, while HPA describes medium consistency with RNA and pending external verification (HPA antibodies; HPA tissue IHC). The payload does not report “Enhanced” validation. Interpret unexpected staining with controls and, where feasible, an independent IHC-approved antibody (general IHC practice). |
| What IF/ICC can establish here | HPA supplies no main ICC-IF location and no cell lines with ICC-IF images; the listed antibodies have no ICC status in the payload (HPA subcellular; HPA antibodies). For the separate IF/ICC guide, ask: “Is a ROCK1 IF pattern validated here?” Answer: “No IF localization pattern is established by these HPA records.” |
| Retrieval and epitope information | No antibody epitope or target-specific retrieval condition is supplied in the UniProt and HPA records. Follow the selected IHC-validated antibody’s documented IHC-P conditions and assess retrieval against a positive control (general IHC practice). These records do not establish that fixation changes ROCK1 staining. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive cells are blank. | The result disagrees with HPA’s high staining in the chosen cells; the reason is unresolved (HPA tissue IHC). | Check cell identity and section quality, then follow the selected antibody’s IHC-P retrieval and dilution instructions. Verify detection with a positive control (general IHC practice). |
| Only nuclei stain in otherwise suitable positive tissue. | Exclusive nuclear signal conflicts with ROCK1’s predominantly cytoplasmic distribution (HPA tissue IHC; UniProt Q13464). | Compare with a no-primary control and an independently IHC-approved ROCK1 antibody; review counterstain and chromogen appearance before scoring (HPA antibodies; general IHC practice). |
| Cardiomyocytes or skeletal myocytes stain strongly. | Those cell types are “Not detected” in HPA tissue IHC; cross-reactivity or endogenous detection activity is possible (HPA tissue IHC; general IHC practice). | Confirm which cells carry the signal. Run appropriate no-primary and detection controls, then compare staining with another IHC-approved antibody (HPA antibodies; general IHC practice). |
| Diffuse brown background obscures cell boundaries. | Nonspecific binding or detection background can conceal the cytoplasmic pattern expected by HPA (HPA tissue IHC; general IHC practice). | Inspect a no-primary control; review blocking, wash steps, antibody dilution and chromogen development under the selected IHC-P workflow (general IHC practice). |
| Signal is weak in a low-staining comparator. | HPA reports low staining in hippocampal or caudate neurons and several other specified cell populations (HPA tissue IHC). | Compare with a high-staining cell population on a suitable control section before changing conditions; a weak low-comparator signal alone is inconclusive (HPA tissue IHC; general IHC practice). |
| An IF image appears to disagree with the IHC pattern. | The supplied HPA subcellular record provides neither a main ICC-IF location nor ICC-IF images for ROCK1 (HPA subcellular). | Keep the IF interpretation separate. Verify cell identity, controls and antibody suitability within the dedicated IF/ICC guide; do not treat this IHC pattern as an IF validation (HPA antibodies; general IF practice). |
Experiment evidence
IHC & IF images showing ROCK1 staining
M00722 · IHCImmunohistochemical analysis of paraffin-embedded human kidney, using ROCK1 Antibody(M00722) ROCK1 was detected in paraffin-embedded tissue
M00722 · IHCImmunohistochemical analysis of paraffin-embedded human kidney, using ROCK1 Antibody(M00722) ROCK1 was detected in paraffin-embedded tissue
M00722 · IFImmunofluorescent analysis of Hela cells, using ROCK1 Antibody .
M00722-2 · IHCHuman testis was stained with anti-ROCK1 rabbit antibody
A00722 · IFImmunocytochemistry of ROCK1 in 293 cells with ROCK1 antibody at 10 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000067900-ROCK1/tissue/cerebral+cortex#imid_2414412
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000067900-ROCK1/tissue/colon#imid_2414354
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000067900-ROCK1/tissue/liver#imid_2414347
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000067900-ROCK1/tissue/kidney#imid_2414340
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ROCK1 IHC & IF
🧪Run colon first; glandular cells should stain (HPA: High in colon glandular cells). Use heart muscle cardiomyocytes as the negative tissue (HPA: Not detected in cardiomyocytes); on the colon slide, treat adjacent nonglandular cells as an internal background reference only if they show no specific staining (standard IHC practice).
Positive control tissue: Appendix (Lymphoid tissue, HPA High)
Negative control tissue: Heart muscle (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for ROCK1; derive a cell-line control from the positive tissue's cell type (Lymphoid tissue) and confirm it by RNA or western blot first.
Technical controls: Include no-primary (secondary-only) and host- and clonality-matched isotype controls, plus a validated ROCK1 knockout specimen or immunizing-peptide block to assess specificity (standard IHC practice). For chromogenic detection, block endogenous peroxidase and assess endogenous biotin background because the reported detection uses a biotin-based complex and DAB (caption: biotinylated secondary, SABC and DAB).
⚠️Feasibility: A ROCK1-specific fixation window or fixation effect is unreported, and the selected M00722 tissue-IHC caption does not state the fixative (caption: fixative unreported). The paraffin-section example used heat retrieval in citrate buffer at pH 6 for 20 minutes, but it does not establish that retrieval is required for every specimen (caption: retrieval conditions). There is no supplied basis to call frozen sections or IF easier (HPA: no ICC-IF images); in colon, check that luminal mucin or pigment is not scored as cellular DAB staining (standard IHC practice).
HPA tissue IHC evidence for ROCK1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Pending external verification.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Appendix | Lymphoid tissue | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | High | Protein (IHC) | HPA → |
Section 3
Advanced ROCK1 IHC Tips
Troubleshoot ROCK1 staining in paraffin sections by checking retrieval, cell compartment, controls, and the limits of the available antibody evidence.
What should I adjust if ROCK1 staining is weak after antigen retrieval?
Use heat-mediated citrate buffer at pH 6 for 20 minutes as the starting retrieval condition for this paraffin-section assay (datasheet M00722). The selected kidney image used that condition, although its fixative was not reported (datasheet M00722). If staining is weak, compare retrieval times around 20 minutes on matched sections while keeping antibody concentration and detection constant (standard IHC practice). Check tissue morphology alongside signal, because excessive heating can damage sections and make staining harder to interpret (standard IHC practice). Include a known positive compartment to distinguish poor retrieval from a genuinely low-expressing region (HPA: cytoplasmic expression in most tissues; standard IHC practice).
Can I infer the best fixative for ROCK1 from the kidney IHC image?
No target-specific fixation sensitivity is established by the supplied evidence: the selected paraffin-embedded kidney caption does not state its fixative (datasheet M00722). Record the fixative, fixation duration, and processing history for each specimen before comparing ROCK1 staining across batches (standard IHC practice). If signal differs between batches, stain matched control sections together and change one preparation variable at a time (standard IHC practice). Do not assign a fixation effect from the reported tissue staining pattern or ROCK1 sequence features; neither provides a direct fixation comparison (HPA: tissue IHC profile; UniProt Q13464: topology and modifications). Preserve morphology when optimizing, since damaged cells can confound compartment-level scoring (standard IHC practice).
Where should ROCK1 staining appear in a convincing tissue section?
Expect mainly cytoplasmic staining across many tissues, while assessing the actual cell type and morphology in each section (HPA: cytoplasmic expression in most tissues). ROCK1 is also annotated at cytoskeletal structures, cell projections, the cell membrane, and a small Golgi-associated fraction (UniProt Q13464: subcellular location). A focal peripheral pattern can therefore be plausible, but diffuse nuclear-only signal needs independent validation before attribution to ROCK1 (UniProt Q13464: subcellular location; standard IHC practice). Compare the suspect region with a positive control and a section processed without primary antibody (standard IHC practice). Interpret fine projections cautiously in chromogenic sections, where overlapping cells can obscure compartment boundaries (standard IHC practice).
Could epitope position explain inconsistent ROCK1 staining?
The supplied record lists 0 annotated isoforms, and the selected antibody caption does not map its epitope (UniProt Q13464: isoforms; datasheet M00722). ROCK1 contains a kinase domain at residues 76–338, a Rho-binding domain at 949–1015, and a PH domain at 1118–1317 (UniProt Q13464: domains). Without an epitope map, do not attribute a weak region to a particular domain or modified residue (UniProt Q13464: domains and modifications; standard IHC practice). Compare retrieval conditions on adjacent sections and evaluate staining with a separately validated antibody if available (standard IHC practice). Document the antibody identifier and processing conditions so discordant results remain traceable (standard IHC practice).
How should I investigate ROCK1 localisation by multiplex immunofluorescence?
Treat IF as a separate validation experiment: the selected antibody evidence describes chromogenic staining of a paraffin kidney section, not an IF result (datasheet M00722). Pair ROCK1 with a validated marker for the cell population being examined, such as a marker appropriate to the macrophages scored high in lung (HPA: high in lung macrophages; standard IF practice). Choose fluorophores after checking tissue autofluorescence in unstained controls, and separate channels sufficiently to assess bleed-through (standard IF practice). ROCK1 has no annotated transmembrane segment, so test permeabilisation for access to its intracellular epitopes; the antibody's exact epitope is unspecified (UniProt Q13464: topology; datasheet M00722). Compare the resulting cellular pattern with the cytoplasmic tissue profile (HPA: tissue IHC profile).
What should I check when ROCK1 DAB staining appears widespread?
First inspect a section processed without primary antibody and check endogenous peroxidase blocking before assigning diffuse DAB signal to ROCK1 (standard IHC practice). The selected image used 10% goat serum blocking, 1 µg/mL primary antibody, biotinylated secondary detection, and DAB (datasheet M00722). If background persists, compare primary concentrations around 1 µg/mL and verify that secondary detection is compatible with the specimen (datasheet M00722; standard IHC practice). Examine folds, section edges, and damaged areas separately from intact tissue because these features can distort chromogenic staining (standard IHC practice). Widespread cytoplasmic signal can be plausible, but intensity alone does not establish specificity (HPA: cytoplasmic expression in most tissues; standard IHC practice).
How should I score ROCK1 staining across tissue regions? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because ROCK1 tissue staining is reported mainly as cytoplasmic (HPA: tissue IHC profile). For a chromogenic comparison, use an H-score or the percentage of positive cells with a prespecified intensity threshold (standard IHC practice). If counting a selected population, report positive cells per mm² and normalise to the number or area of eligible cells in that region (standard IHC practice). Keep retrieval, antibody concentration, detection, imaging, and threshold settings consistent across comparison groups (standard IHC practice). Exclude folds, necrotic areas, and poorly preserved regions by predefined rules, and report those exclusions with the scores (standard IHC practice).
How can I distinguish genuine ROCK1 staining from an artefact?
A plausible positive signal should fit the cell morphology and predominantly cytoplasmic tissue pattern reported for ROCK1 (HPA: tissue IHC profile). High staining in kidney glomerular cells or lung macrophages is supported by tissue observations, whereas cardiomyocytes and skeletal myocytes were reported as not detected (HPA: tissue IHC). Treat isolated nuclear-only staining, strong section-edge signal, and staining in necrotic areas as findings requiring control review (UniProt Q13464: subcellular location; standard IHC practice). Check the section without primary antibody and the peroxidase control for detection-related colour before scoring suspect cells (standard IHC practice). HPA labels its tissue staining Approved with medium consistency and pending external verification, so report discordant patterns with that limitation (HPA: reliability).
Boster reagents
Best ROCK1 / Rho-associated protein kinase 1 IHC Antibodies
The catalog includes ROCK1 antibodies with IHC images from human kidney and testis, rat tissues, and an IF/ICC image from 293 cells (catalog image captions).
Real IHC data
Anti-ROCK1 Rabbit Monoclonal Antibody
Cat # M00722
Real IHC data
Anti-ROCK1 Rabbit Monoclonal Antibody
Cat # M00722-2
Real IF data
Anti-ROCK1 Antibody
Cat # A00722
M00722 has IHC images from human kidney paraffin sections and rat tissues (M00722 image captions); M00722-2 has an IHC image from human testis (M00722-2 image caption). A00722 has an ICC image from 293 cells and lists IF and ICC applications (A00722 image captions and applications).
Which to pick: For tissue IHC, choose M00722 when a paraffin-section example is useful: its human kidney caption reports citrate retrieval at pH 6 for 20 minutes and 10% goat serum blocking; the fixative is unreported (M00722 image caption). For IF/ICC, choose A00722 for its 293-cell images, including IF at 20 μg/mL (A00722 image captions). For work spanning human and rat tissues, M00722 has IHC images in both species; mouse reactivity is listed, but no mouse IHC image is supplied (M00722 image captions and reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q13464 (ROCK1_HUMAN, Rho-associated protein kinase 1).
- Human Protein Atlas. ROCK1 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ROCK1 subcellular location (ICC-IF): Highest expression in REH: 11.3 nTPM.
- Human Protein Atlas. ROCK1 antibody validation summary (3 antibodies).
- Up regulation of Rho-associated coiled-coil containing kinase1 (ROCK1) is associated with genetic instability and poor prognosis in prostate cancer. Aging 2019 — PMC6781985.
- Molecular insights into treatment-resistant erectile dysfunction: staining patterns of human corpora cavernosa-a preliminary study. Translational andrology and urology 2025 — PMC12683496.
- MicroRNA-1291 promotes endometrial fibrosis by regulating the ArhGAP29-RhoA/ROCK1 signaling pathway in a murine model. Molecular medicine reports 2017 — PMC5647010.
- Investigation of Rho-Kinase Expressions and Polymorphisms in Mantle Cell Lymphoma Patients. Turkish journal of haematology : official journal of Turkish Society of Haematology 2016 — PMC5100726.
- PubMed PMID:8617235 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:9722579 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ROCK1 (Rho-associated protein kinase 1). Expected localisation Predominantly cytoplasmic in tissue sections (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet M00722). Validated IHC antibody: M00722 at 1:50 (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Lymphoid tissue); negative Heart muscle. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human kidney tissue; fixative not specified (datasheet M00722); 2. Fixation: Image fixative and duration unreported (datasheet M00722); 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 M00722); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M00722); 8. Primary antibody: Rabbit monoclonal (clone CDE-18) anti-ROCK1, 1:50 (datasheet M00722); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); ROCK1-specific effects are unknown.; Caveat: Staining varies by cell type within tissues (HPA tissue IHC); Isoform / epitope: No annotated isoforms or extracellular segment (UniProt); Regulation: No expression regulator established (UniProt). Evidence: UniProt Q13464, HPA tissue IHC · Approved, HPA cell-line RNA · Highest expression in REH: 11.3 nTPM, Datasheet M00722, PMC6781985, PMC12683496. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ROCK1/immunohistochemistry-ihc-guide.