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RHOT1 / Mitochondrial Rho GTPase 1 · IHC design guide
Design Immunohistochemistry for RHOT1
Plan RHOT1 chromogenic IHC around granular cytoplasmic tissue staining (HPA tissue IHC) and its outer mitochondrial membrane location (UniProt). The catalog antibody has a human paraffin-section starting range of 2–5 μg/mL (datasheet: A05928-1).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RHOT1 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 | Observed granular cytoplasm (HPA tissue IHC); outer mitochondrial membrane (UniProt) | |
| Staining pattern | Granular cytoplasmic staining across diverse cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A05928-1) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Broad expression can complicate cell-specific scoring (HPA tissue IHC) | |
| Regulation | No stimulus-linked regulator specified (UniProt) | |
| Isoform / epitope | 7 isoforms; epitope differences unspecified (UniProt) |
Section 1
Recommended RHOT1 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet A05928-1) with published RHOT1 tissue IHC methods (PMC8973856; PMC3409151).
| Sample | Paraffin-embedded human thyroid papillary carcinoma tissue; fixative not specified (datasheet A05928-1) |
| Fixation | Image fixative and duration unreported (datasheet A05928-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A05928-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A05928-1) |
| Primary antibody | Rabbit anti-RHOT1, 2-5 μg/ml (datasheet A05928-1) |
| Primary incubation | Overnight at 4 °C (datasheet A05928-1) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A05928-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | RHOT1-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression with a granular pattern. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (datasheet A05928-1); the pancreatic TMA method used citrate at pH 6 (PMC3409151).
Section 2
What Is the Expected RHOT1 Staining Pattern?
RHOT1 is an outer mitochondrial membrane protein with a cytoplasmic domain spanning residues 1–592 (UniProt Q8IXI2 topology). In paraffin sections, expect granular cytoplasmic staining, including in the glandular and epithelial cells reported as highly stained by HPA (HPA: tissue IHC). HPA rates the tissue pattern Supported, based on consistency between antibody staining and RNA expression (HPA: reliability).
| Granular cytoplasmic signal in adrenal glandular cells or bronchial respiratory epithelial cells. | This matches the reported RHOT1 tissue pattern and High staining in these cells (HPA: tissue IHC). Its cytoplasmic appearance is consistent with an outer mitochondrial membrane protein (UniProt Q8IXI2: subcellular location and topology). Assess the cell type and pattern together; color alone does not establish specificity (general IHC practice). |
| Predominantly nuclear staining, with little granular cytoplasmic signal. | A nuclear dominant pattern conflicts with HPA’s general granular cytoplasmic profile and UniProt’s outer mitochondrial membrane assignment (HPA: tissue IHC; UniProt Q8IXI2: subcellular location). Treat it as a possible artifact and check controls and the detection workflow before interpreting it as RHOT1 (general IHC practice). |
| Strong staining in a cell population outside the expected pattern for that section. | Check the cell identity against the relevant HPA tissue image before calling the signal specific (HPA: tissue IHC). Unexpected staining can reflect cross-reactivity or endogenous detection activity (general IHC practice), but RHOT1 is broadly expressed (UniProt Q8IXI2: tissue specificity); staining in an additional cell type alone does not prove an artifact. |
| Uniform haze across tissue structures or a broadly colored slide background. | HPA describes a granular cytoplasmic pattern, so uniform haze is a poor match (HPA: tissue IHC). It can arise from nonspecific antibody binding, insufficient washing, or detection background (general IHC practice). Compare the haze with a negative reagent control before scoring faint cytoplasmic color as RHOT1 (general IHC practice). |
| No visible staining in adrenal glandular cells or bronchial respiratory epithelial cells. | Both are reported as High staining populations, making absence of signal a reason to investigate the run (HPA: tissue IHC). Check tissue preservation, retrieval, antibody application, and detection controls using the chosen assay’s instructions (general IHC practice). HPA’s tissue data do not identify which step caused the failure. |
💡Expected RHOT1 appearanceCall a positive result when the appropriate cells show discernible granular cytoplasmic staining, potentially strong in HPA High populations (HPA: tissue IHC); isolated nuclear color or uniform background is discordant with that profile (HPA: tissue IHC; general IHC practice).
| Which tissue and cells anchor interpretation? | Use an HPA High population, such as breast glandular cells, caudate neuronal cells, or cervix squamous epithelial cells, as a pattern reference (HPA: tissue IHC). HPA lists no negative or low examples in the supplied record, so it does not support designating a particular tissue as RHOT1 negative. |
| Topology and antibody epitope | RHOT1 has one transmembrane segment at 593–615, a cytoplasmic region at 1–592, and a short intermembrane region at 616–618 (UniProt Q8IXI2: topology). Epitope position could matter when assessing staining, but no epitope for the antibody used here is supplied; do not infer its accessibility or a retrieval requirement. |
| Isoforms and antibody validation | UniProt lists 7 RHOT1 isoforms (UniProt Q8IXI2: isoforms). Their recognition by a particular antibody is unspecified. HPA lists IHC as Supported for HPA010687, CAB068223, and CAB068224 (HPA: antibodies); that status does not establish identical isoform coverage or staining behavior for every antibody. |
| Endogenous detection activity | In chromogenic IHC, endogenous enzyme activity or tissue pigment may resemble weak signal, depending on the detection system (general IHC practice). Interpret questionable color against the appropriate detection controls and cellular pattern (general IHC practice). The supplied HPA and UniProt records do not identify a RHOT1 specific endogenous detection effect. |
| IF/ICC: what pattern is established? | HPA’s subcellular summary says “Membrane,” but gives no main location or ICC/IF image bearing cell lines; the listed antibodies have no ICC status in this payload (HPA: subcellular; HPA: antibodies). UniProt assigns RHOT1 to the outer mitochondrial membrane (UniProt Q8IXI2: subcellular location). These records do not establish an antibody validated IF/ICC staining pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| The known High cell population is blank. | The run may have failed at staining or detection; HPA does not identify a target specific failure mechanism (HPA: tissue IHC). | Verify section identity and positive control, then review retrieval, primary antibody application, and detection according to the assay instructions (general IHC practice). |
| Color is chiefly nuclear. | This conflicts with the reported granular cytoplasmic pattern and mitochondrial location (HPA: tissue IHC; UniProt Q8IXI2: subcellular location). | Check a negative reagent control, counterstain, and detection background before scoring the color as RHOT1 (general IHC practice). |
| Signal is diffuse rather than granular. | Diffuse color does not match HPA’s general granular cytoplasmic profile (HPA: tissue IHC); nonspecific binding or excess background is possible (general IHC practice). | Compare the negative reagent control and review blocking, antibody concentration, washing, and chromogen development (general IHC practice). |
| Unexpected cells stain strongly. | Cross-reactivity or endogenous detection activity is possible (general IHC practice), although RHOT1 is broadly expressed (UniProt Q8IXI2: tissue specificity). | Identify the cell population, compare its HPA tissue pattern, and check detection controls before assigning specificity (HPA: tissue IHC; general IHC practice). |
| Two antibodies give different patterns. | The listed antibodies are IHC Supported, but their epitopes and isoform coverage are unspecified here (HPA: antibodies; UniProt Q8IXI2: isoforms). | Compare staining in the same HPA reported cell population and review each antibody’s documented assay conditions; do not assume matching epitopes (HPA: tissue IHC; general IHC practice). |
| IF/ICC fluorescence differs from the IHC pattern. | HPA supplies a membrane summary but no ICC/IF images or ICC validation status for the listed antibodies (HPA: subcellular; HPA: antibodies). | Treat the fluorescence pattern as unestablished by these sources; assess it with appropriate imaging controls and the separate IF/ICC guide (general IF practice). |
Experiment evidence
IHC & IF images showing RHOT1 staining
A05928-1 · IHCIHC analysis of MIRO1/RHOT1 using anti-MIRO1/RHOT1 antibody (A05928-1). MIRO1/RHOT1 was detected in a paraffin-embedded section of human thy
A05928-1 · IHCIHC analysis of MIRO1/RHOT1 using anti-MIRO1/RHOT1 antibody (A05928-1). MIRO1/RHOT1 was detected in a paraffin-embedded section of human gal
A05928-1 · IFIF analysis of MIRO1/RHOT1 using anti-MIRO1/RHOT1 antibody (A05928-1). MIRO1/RHOT1 was detected in an immunocytochemical section of T47D cel
A05928 · IHCImmunohistochemistry of RHOT in mouse brain tissue with RHOT antibody at 5 μg/ml.
A05928 · IFImmunofluorescence of RHOT in mouse brain tissue with RHOT antibody at 20 μg/ml.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000126858-RHOT1/tissue/cerebral+cortex#imid_2879864
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000126858-RHOT1/tissue/colon#imid_2879798
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000126858-RHOT1/tissue/liver#imid_2879791
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000126858-RHOT1/tissue/kidney#imid_2879784
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RHOT1 IHC & IF
🧪Run adrenal gland first; its glandular cells should stain (HPA: High in adrenal gland glandular cells). HPA detects RHOT1 in all 45 scored tissues, so there is no supported negative tissue or confirmed negative cell population within this slide; use the no-primary and isotype slides to judge background in glandular and surrounding cells (HPA: no negative rows; RHOT1 detected in all 45 scored tissues).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: RHOT1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for RHOT1; derive a cell-line control from the positive tissue's cell type (Glandular cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only slide and a concentration-matched rabbit IgG isotype control (selected-SKU IHC caption: rabbit primary antibody); use RHOT1 knockout material, if available, as a biological specificity control (standard IHC practice). For the caption’s biotin-based DAB detection, block endogenous peroxidase and assess endogenous biotin and native pigment on the adrenal section (selected-SKU IHC caption: biotinylated secondary, SABC and DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU IHC caption: fixative not stated). Heat retrieval in EDTA at pH 8.0 is a documented starting condition, but its necessity for adrenal tissue has not been established here (selected-SKU IHC caption: thyroid papillary carcinoma section with EDTA retrieval). No supplied comparison shows frozen sections or IF to be easier; for adrenal DAB sections, assess native pigment alongside the expected mitochondrial pattern (HPA: no ICC-IF cell-line images; UniProt Q8IXI2: mitochondrial outer membrane; standard IHC practice).
HPA tissue IHC evidence for RHOT1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — High consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: RHOT1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced RHOT1 IHC Tips
Troubleshoot RHOT1 staining by checking retrieval, compartment, controls, and scoring against the evidence available for paraffin section IHC.
Which retrieval condition should I try first for RHOT1 paraffin sections?
Start with heat mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A05928-1). The selected image used that condition before overnight incubation at 4°C with 2 μg/ml antibody, so keep those steps consistent while assessing retrieval (datasheet A05928-1). Compare staining in matched sections with and without retrieval, judging whether granular cytoplasmic signal improves without a parallel rise in diffuse background (HPA tissue IHC; standard IHC practice). If signal remains weak, test a different retrieval buffer on adjacent sections as a fallback, with the primary antibody concentration and detection held constant (standard IHC practice).
How should I troubleshoot fixation when RHOT1 staining is weak?
The selected RHOT1 tissue caption identifies a paraffin section but does not report its fixative, so target specific fixation sensitivity is unknown (datasheet A05928-1). Record the fixative, fixation duration, tissue thickness, and processing history for each specimen before comparing signal across cases (standard IHC practice). Assess matched sections with the same EDTA pH 8.0 retrieval and 2 μg/ml primary incubation to isolate processing differences (datasheet A05928-1; standard IHC practice). Compare intact and poorly preserved regions for granular cytoplasmic staining, and avoid interpreting a fixation dependent change as altered RHOT1 abundance without a matched processing control (HPA tissue IHC; standard IHC practice).
What RHOT1 staining pattern is plausible in tissue sections?
Expect cytoplasmic staining with a granular pattern in tissue IHC, consistent with RHOT1 on the mitochondrial outer membrane (HPA tissue IHC; UniProt Q8IXI2). Its residues 1–592 face the cytoplasm, while a membrane segment spans residues 593–615 and the short terminal segment faces the intermembrane space (UniProt Q8IXI2 topology). Review signal within morphologically intact cells rather than treating every brown deposit as mitochondrial staining, because chromogenic IHC alone does not resolve individual mitochondrial membranes (standard IHC practice). Predominantly nuclear or uniformly extracellular staining needs additional specificity checks, including a negative reagent control and comparison with the expected granular pattern (HPA tissue IHC; standard IHC practice).
Could isoforms or epitope accessibility explain discordant RHOT1 staining?
RHOT1 has 7 listed isoforms, so an antibody's epitope and its presence across isoforms matter when comparing specimens or antibodies (UniProt Q8IXI2). The supplied tissue caption reports the antibody concentration and retrieval but gives no epitope coordinates or isoform coverage; do not assign a negative section to a particular isoform from this evidence (datasheet A05928-1). The protein includes Miro regions, 2 EF hands, and a membrane anchor near its C terminus, which can guide questions for an antibody supplier about epitope placement (UniProt Q8IXI2). Compare serial sections and, where available, independently mapped antibodies before attributing discordance to epitope masking or alternative splicing (standard IHC practice).
How can IF help assess the RHOT1 pattern seen by IHC?
For a separate IF experiment, multiplex RHOT1 with a validated marker for the cell type being evaluated and a mitochondrial marker to assess cellular identity and organelle association (UniProt Q8IXI2; standard IF practice). Choose fluorophores after checking unstained tissue for autofluorescence, favoring a channel with lower background and including single stain controls for spectral bleed through (standard IF practice). Plan permeabilisation around the antibody's mapped epitope: residues 1–592 are cytoplasmic, whereas residues 616–618 face the mitochondrial intermembrane space (UniProt Q8IXI2 topology). Because the supplied IHC caption gives neither an IF fixation method nor an epitope location, establish IF access and specificity with matched controls (datasheet A05928-1; standard IF practice).
How do I reduce diffuse brown signal without losing RHOT1 staining?
Begin with a no primary control, a peroxidase block, and inspection of endogenous pigment before assigning brown signal to RHOT1 (standard IHC practice). The selected tissue workflow used 10% goat serum, 2 μg/ml primary antibody overnight at 4°C, a biotinylated secondary for 30 minutes at 37°C, and DAB detection (datasheet A05928-1). If background persists, titrate the primary and strengthen washing or blocking one variable at a time while monitoring expected granular cytoplasmic signal (HPA tissue IHC; standard IHC practice). With a biotin based detection system, compare an appropriate detection control when tissue associated background is suspected (datasheet A05928-1; standard IHC practice).
How should RHOT1 IHC be scored across heterogeneous tissue regions? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and viable tissue region before scoring, and apply the same threshold and imaging conditions to every section (standard IHC practice). For cell level comparison, report the percentage of positive cells and an H-score based on staining intensity; for spatial questions, report positive cell density per mm² of viable tissue (standard IHC practice). Score granular cytoplasmic signal within cells, consistent with the reported tissue pattern, and record diffuse staining separately (HPA tissue IHC; standard IHC practice). Normalize counts to the relevant cell population or viable area, then compare cases only when section processing, retrieval, antibody concentration, and detection are matched (standard IHC practice).
What separates credible RHOT1 staining from section artefacts?
Credible RHOT1 signal should appear as cytoplasmic granularity in intact cells, consistent with the tissue IHC profile and mitochondrial outer membrane location (HPA tissue IHC; UniProt Q8IXI2). Examine the annotated cell type rather than equating signal in an adjacent cell population with expression in the cells under study (standard IHC practice). Treat staining concentrated at section edges, necrotic areas, or damaged folds cautiously, and check whether similar deposits remain in the no primary control (standard IHC practice). A positive peroxidase or detection control signal without primary antibody calls for resolving endogenous enzyme or detection background before interpreting DAB intensity as RHOT1 abundance (standard IHC practice).
Boster reagents
Best RHOT1 / Mitochondrial Rho GTPase 1 IHC Antibodies
Two anti-RHOT1 antibodies have real IHC images from human paraffin sections and mouse brain, plus IF images from T47D cells and mouse brain (catalog image captions).
Real IHC data
Anti-MIRO1/RHOT1 Antibody ®
Cat # A05928-1
Real IHC data
Anti-Mitochondrial Rho GTPase 1 RHOT1 Antibody
Cat # A05928
A05928-1 has IHC images from human paraffin sections and an IF/ICC image from T47D cells (A05928-1 image captions). A05928 has IHC and IF images from mouse brain (A05928 image captions).
Which to pick: For human paraffin-section IHC, choose A05928-1; its thyroid papillary carcinoma image used EDTA retrieval at pH 8.0 and 2 μg/ml antibody, with the fixative unreported (A05928-1 IHC caption). For IF/ICC, A05928-1 has a T47D-cell image at 5 μg/mL; A05928 has a mouse-brain IF image at 20 μg/mL (respective IF captions). For mouse tissue IHC, A05928 has a mouse-brain image at 5 μg/mL, with processing and fixative unreported; both antibodies list Human, Mouse, and Rat reactivity, but their images document only the stated samples, and neither has a specified clone (A05928 IHC caption; catalog reactivity and clone fields).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8IXI2 (MIRO1_HUMAN, Mitochondrial Rho GTPase 1).
- Human Protein Atlas. RHOT1 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. RHOT1 subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. RHOT1 antibody validation summary (3 antibodies).
- Hsa_circ_0005230 is up-regulated and promotes gastric cancer cell invasion and migration via regulating the miR-1299/RHOT1 axis. Bioengineered 2022 — PMC8973856.
- RhoT1 and Smad4 are correlated with lymph node metastasis and overall survival in pancreatic cancer. PloS one 2012 — PMC3409151.
- PubMed PMID:12696059 — UniProt-cited evidence.
- PubMed PMID:12482879 — UniProt-cited evidence.
- PubMed PMID:11230166 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RHOT1 (Mitochondrial Rho GTPase 1). Expected localisation Observed granular cytoplasm (HPA tissue IHC); outer mitochondrial membrane (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A05928-1). Validated IHC antibody: A05928-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human thyroid papillary carcinoma tissue; fixative not specified (datasheet A05928-1); 2. Fixation: Image fixative and duration unreported (datasheet A05928-1); 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: EDTA pH 8.0 (datasheet A05928-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A05928-1); 8. Primary antibody: Rabbit anti-RHOT1, 2-5 μg/ml (datasheet A05928-1); 9. Primary incubation: Overnight at 4 °C (datasheet A05928-1). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Broad expression can complicate cell-specific scoring (HPA tissue IHC); Isoform / epitope: 7 isoforms; epitope differences unspecified (UniProt); Regulation: No stimulus-linked regulator specified (UniProt). Evidence: UniProt Q8IXI2, HPA tissue IHC · Supported, HPA ICC-IF · Membrane, Datasheet A05928-1, PMC8973856, PMC3409151. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RHOT1/immunohistochemistry-ihc-guide.