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LMO2 / Rhombotin-2 · IHC design guide
Design Immunohistochemistry for LMO2
Plan LMO2 paraffin-section IHC around nuclear staining in bone marrow hematopoietic cells and cytoplasmic staining in several tissues (HPA tissue IHC). Use the tissue pattern and the reported staining uncertainty when selecting controls and scoring results (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
LMO2 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 | Nuclear in marrow; cytoplasmic in several tissues (HPA tissue IHC) | |
| Staining pattern | Marrow hematopoietic nuclei; cytoplasm in several tissues (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet M03502) | |
| Positive control | Adrenal gland+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 | Presumed off-target staining; verification pending (HPA tissue IHC) | |
| Regulation | Expression regulators are not specified (UniProt) | |
| Isoform / epitope | 3 isoforms; epitope differences unreported (UniProt) |
Section 1
Recommended LMO2 IHC & IF Protocols
The catalog antibody has an IHC-P protocol (datasheet: M03502). The published protocols below cover lymph node, bone marrow, and whole tissue or microarray sections (PMC8234657; PMC9427670; PMC4305438).
| Sample | Paraffin-embedded lymph node of rat lung tissue; fixative not specified (datasheet M03502) |
| Fixation | Image fixative and duration unreported (datasheet M03502); 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 M03502); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M03502) |
| Primary antibody | Rabbit monoclonal (clone ACOA-12) anti-LMO2, 1:50 (datasheet M03502) |
| Primary incubation | Overnight at 4 °C (datasheet M03502) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet M03502) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | LMO2-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Nuclear expression in bone marrow and cytoplasmic expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (datasheet: M03502). Published protocols used high-pH or mild retrieval with different antibodies (PMC8234657; PMC4305438).
Section 2
What Is the Expected LMO2 Staining Pattern?
LMO2 is a nuclear protein with no transmembrane segment or signal peptide (UniProt P25791). In paraffin-section IHC, expect strong nuclear staining in bone marrow hematopoietic cells (HPA tissue IHC: High; nuclear expression). HPA also reports cytoplasmic expression in several tissues; its tissue IHC reliability is Approved, with presumed off-target binding disregarded and external verification pending (HPA tissue IHC). Interpret staining by cell type and compartment together.
| Distinct, strong staining of hematopoietic cell nuclei in bone marrow, with nuclei identifiable by counterstain. | This is the clearest tissue-level positive pattern in the supplied record: hematopoietic cells are High, and the bone marrow pattern is nuclear (HPA tissue IHC). Nuclear localisation agrees with UniProt P25791. Record the fraction of stained cells as well as intensity; intensity alone can obscure whether the expected cells carry the signal (general IHC practice). |
| Predominantly cytoplasmic staining, with little or no convincing nuclear signal. | Treat this as uncertain LMO2 localisation, especially in bone marrow, where HPA describes nuclear expression (HPA tissue IHC; UniProt P25791). HPA also describes cytoplasmic expression in several tissues and notes presumed off-target binding, so cytoplasmic colour alone cannot establish which signal represents LMO2 (HPA tissue IHC). Compare compartments within the same cell type and seek independent validation before scoring it as specific. |
| Strong staining in adipocytes, smooth muscle cells or oral mucosal squamous epithelial cells. | These cell types are recorded as Not detected, making them useful comparisons for unexpected staining, not absolute biological negatives (HPA tissue IHC). Check whether colour persists without primary antibody and whether it tracks tissue structures; cross-reactivity or detection activity becomes more plausible if the expected positive-cell pattern is absent (general IHC practice). |
| Diffuse colour across cells and extracellular space, without crisp cellular boundaries. | This pattern cannot support a nuclear LMO2 call: UniProt places LMO2 in the nucleus, and HPA describes nuclear bone marrow staining (UniProt P25791; HPA tissue IHC). Uneven reagent distribution, excessive detection signal or inadequate washing can create background in chromogenic IHC (general IHC practice). Judge specificity only after the diffuse component is reduced. |
| No visible staining in bone marrow hematopoietic cells. | This conflicts with the High, nuclear bone marrow pattern reported by HPA, but a blank slide does not establish true absence of LMO2 (HPA tissue IHC). First confirm that hematopoietic cells are present and assess whether the staining run worked on its controls (general IHC practice). HPA's Approved designation remains pending external verification (HPA tissue IHC). |
💡Expected LMO2 appearanceCall the tissue-IHC positive pattern when bone marrow hematopoietic nuclei show distinct, strong staining (HPA tissue IHC: High; nuclear expression), consistent with nuclear LMO2 (UniProt P25791); isolated diffuse colour or staining confined to cells HPA lists as Not detected is suspect and needs control-based review (HPA tissue IHC; general IHC practice).
| Compartment and tissue context | UniProt assigns LMO2 to the nucleus, while HPA reports nuclear bone marrow staining and cytoplasmic expression in several tissues (UniProt P25791; HPA tissue IHC). Use the marrow nuclear pattern as the clearest reference; do not assume every HPA High tissue shares it. |
| Strength of tissue evidence | The tissue IHC profile is Approved, but HPA says presumed off-target binding was disregarded and external verification is pending (HPA tissue IHC). The IHC antibody CAB016258 is Approved; the supplied antibody list gives no Enhanced IHC status (HPA antibodies). Treat unexpected compartments as provisional. |
| Protein architecture and variants | LMO2 has two LIM zinc-binding domains and three listed isoforms; the supplied record does not locate this assay's epitope within them (UniProt P25791). Isoform-specific staining differences therefore cannot be predicted from these data. |
| Processing and topology | UniProt lists no signal peptide, propeptide, transmembrane segment or glycosylation sites, and a single chain spanning residues 1–158 (UniProt P25791). These annotations support interpretation as a non-membrane protein; they do not establish antigen-retrieval needs or fixation sensitivity. |
| Situation | Likely cause | Next action |
|---|---|---|
| Bone marrow nuclei are blank or much weaker than expected. | The result conflicts with HPA's High nuclear staining in hematopoietic cells (HPA tissue IHC). A failed staining run, unsuitable tissue section or missed target-cell population are practical possibilities (general IHC practice). | Verify hematopoietic cells on the counterstained section, inspect run controls, and check that retrieval, antibody use and detection followed the chosen IHC-P assay instructions (general IHC practice). Do not assign an LMO2-specific retrieval or fixation effect from this record. |
| Colour appears only in cytoplasm. | HPA reports cytoplasmic expression in several tissues but nuclear expression in bone marrow, and notes presumed off-target binding (HPA tissue IHC). Cytoplasmic-only colour therefore has uncertain specificity. | Record tissue and cell type before scoring. Compare bone marrow nuclear staining and a no-primary control, then seek independent confirmation for a cytoplasmic LMO2 claim (HPA tissue IHC; general IHC practice). |
| Cells expected to be unstained show strong colour. | HPA lists adipocytes, smooth muscle cells and oral mucosal squamous epithelial cells as Not detected (HPA tissue IHC). Cross-reactivity or background is possible, though Not detected does not guarantee universal absence. | Compare the suspect cells with nearby expected-positive cells and a no-primary control; lower nonspecific staining through standard blocking and washing checks before interpreting the result (general IHC practice). |
| Diffuse colour obscures nuclei and cell boundaries. | The slide cannot resolve the reported nuclear bone marrow pattern (HPA tissue IHC). Background from the detection workflow is a general IHC possibility (general IHC practice). | Review reagent coverage, blocking, washes and detection development with run controls; reassess only when nuclei and cell boundaries can be distinguished (general IHC practice). |
| Colour persists in a no-primary-antibody control. | Primary-antibody-independent colour can arise from endogenous detection activity or other background in chromogenic IHC (general IHC practice). That control cannot identify LMO2. | Address endogenous activity with a detection-system-appropriate block, check secondary and chromogen controls, and repeat the comparison with primary antibody (general IHC practice). |
| Q: What should an IF/ICC image show? | HPA's ICC-IF summary places LMO2 mainly in the nucleoplasm, with an Approved main location; three listed antibodies have Approved ICC status (HPA subcellular; HPA antibodies). | A: Look for nucleoplasmic signal and compare it with nuclear counterstain (HPA subcellular; general IF practice). Use the separate IF/ICC guide for its workflow; the IHC tissue pattern alone does not validate an IF/ICC staining run. |
Experiment evidence
IHC & IF images showing LMO2 staining
M03502 · IHCIHC analysis of LMO2 using anti-LMO2 antibody (M03502). LMO2 was detected in a paraffin-embedded section of lymph node of rat lung tissue. H
M03502 · IHCIHC analysis of LMO2 using anti-LMO2 antibody (M03502). LMO2 was detected in a paraffin-embedded section of lymph node of rat lung tissue. H
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000135363-LMO2/tissue/cerebral+cortex#imid_4223880
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000135363-LMO2/tissue/colon#imid_4223822
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000135363-LMO2/tissue/liver#imid_4223823
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000135363-LMO2/tissue/kidney#imid_4223824
HPA · ICC-IF REHHuman Protein Atlas immunofluorescence, REH, antibody HPA029285; green = LMO2, blue = nucleus, red = microtubules.
HPA · ICC-IF REHHuman Protein Atlas immunofluorescence, REH, antibody HPA029285; green = LMO2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for LMO2 IHC & IF
🧪Run bone marrow first and score nuclear staining in hematopoietic cells (HPA: High in bone marrow hematopoietic cells; UniProt P25791: nucleus). Use adipose tissue as the negative tissue, focusing on adipocytes (HPA: Not detected in adipocytes); internal negative cells on the bone marrow slide should show counterstain without nuclear chromogen, but their identity and negativity must be verified.
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show LMO2 in REH, HEL, MCF-7, U2OS, Rh30, with annotated localisation: Nucleoplasm (approved) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and concentration-matched rabbit isotype controls alongside a biological negative such as LMO2-knockout material or a validated peptide-block control (selected M03502 tissue-IHC caption: rabbit primary). Quench endogenous peroxidase in bone marrow before HRP/DAB detection to check for background from hematopoietic cells (HPA: High in bone marrow hematopoietic cells; selected M03502 tissue-IHC caption: HRP/DAB detection).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the fixative is unreported in the selected M03502 tissue-IHC caption. That paraffin-section caption uses heat-mediated EDTA retrieval at pH 8.0, but does not establish that retrieval is required under other conditions (selected M03502 tissue-IHC caption). No supplied comparison establishes whether frozen sections or IF/ICC are easier; for bone marrow chromogenic IHC, endogenous peroxidase is a practical background concern (HPA: High in bone marrow hematopoietic cells; selected M03502 tissue-IHC caption: HRP/DAB detection).
HPA tissue IHC evidence for LMO2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Presumed off target binding observed and disregarded. 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in molecular layer | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Oral mucosa | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Ovary | Ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced LMO2 IHC Tips
Troubleshoot LMO2 staining in paraffin sections by checking retrieval, compartment, cell identity, controls and scoring before interpreting a chromogenic signal.
What retrieval conditions should I start with for LMO2 paraffin-section IHC?
Start with heat-mediated retrieval in EDTA pH 8.0 for LMO2 paraffin-section IHC (datasheet M03502). The selected image used this retrieval before overnight incubation at 4°C with antibody at 1:50 (datasheet M03502); it does not report a retrieval duration (datasheet M03502). If staining is weak, compare retrieval duration on adjacent sections while keeping antibody dilution and DAB development consistent (standard IHC practice). Judge improvement by crisp nuclear staining in appropriate cells rather than by overall brown intensity, because LMO2 is nuclear (UniProt P25791) and HPA reports nuclear expression in bone marrow (HPA: tissue IHC profile).
Could fixation be responsible for weak or uneven LMO2 staining?
The selected paraffin-section caption does not state its fixative, so target-specific LMO2 fixation sensitivity is unknown (datasheet M03502). Record fixative, fixation duration and section age for each specimen, then compare sections processed together under the same EDTA pH 8.0 retrieval and 1:50 antibody conditions used for the catalog image (datasheet M03502; standard IHC practice). If one processing batch stains poorly, include a previously staining control section in the same run to separate processing variation from detection failure (standard IHC practice). Do not attribute weak staining to a particular fixation effect without a controlled comparison (standard IHC practice).
How should I assess nuclear versus cytoplasmic LMO2 staining?
Prioritise a distinct nuclear signal when assessing LMO2, which UniProt places in the nucleus and HPA places mainly in the nucleoplasm (UniProt P25791; HPA: subcellular). HPA also reports nuclear expression in bone marrow and cytoplasmic expression in several tissues, so document compartment and tissue instead of treating every cytoplasmic signal alike (HPA: tissue IHC profile). Compare the DAB pattern with counterstained nuclei at matched magnification and development conditions, including a no-primary control for diffuse colour (standard IHC practice). Because HPA notes presumed off-target binding and pending external verification, require concordant cellular context before accepting an unexpected compartment (HPA: tissue IHC reliability).
Can an isoform or masked epitope explain discrepant LMO2 IHC results?
LMO2 has 3 listed isoforms and 2 LIM zinc-binding domains, spanning residues 30–89 and 94–153 in the recorded sequence (UniProt P25791). The supplied caption gives no antibody epitope, so it cannot establish which isoforms the catalog antibody detects in sections (datasheet M03502). When results disagree, check the antibody’s documented immunogen or epitope before claiming isoform selectivity, and compare sections using the same EDTA pH 8.0 retrieval (standard IHC practice; datasheet M03502). LMO2 has no annotated glycosylation or modified-residue sites in this record; those annotations alone cannot explain a discrepant staining pattern (UniProt P25791).
How can I check LMO2 localisation by multiplex IF?
Use IF as a separate localisation check: HPA reports LMO2 mainly in the nucleoplasm and lists ICC/IF images, including REH and HEL (HPA: subcellular). Multiplex LMO2 with a marker for the cell population being evaluated, and choose a far-red detection channel when tissue autofluorescence obscures shorter-wavelength signal (standard IF practice). LMO2 has no transmembrane segment and is annotated as nuclear, so permeabilisation must permit antibody access to the nucleus (UniProt P25791; standard IF practice). Titrate permeabilisation against morphology and background, include single-stain controls for channel bleed-through, and do not transfer the IHC 1:50 dilution to IF without validation (standard IF practice; datasheet M03502).
What should I adjust when LMO2 DAB staining has high background?
The selected paraffin-section image used 10% goat serum blocking, a 1:50 primary antibody overnight at 4°C, and a peroxidase-linked detection system with DAB (datasheet M03502). Start by checking a no-primary section, endogenous peroxidase blocking, wash quality and DAB development time; these are general chromogenic IHC controls (standard IHC practice). If background persists, titrate the primary antibody around the documented condition while holding retrieval at EDTA pH 8.0 and comparing adjacent sections (datasheet M03502; standard IHC practice). Retain conditions that distinguish cellular signal from diffuse staining, especially because HPA reports presumed off-target binding in its tissue assessment (HPA: tissue IHC reliability).
How should I quantify LMO2 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue region and cell population before scoring, then report the percentage of LMO2-positive nuclei and staining intensity separately (standard IHC practice; UniProt P25791 nucleus). An H-score combines intensity grades 0–3 with their percentages to give a 0–300 score; positive-cell density per mm² is useful when cell abundance varies (standard IHC practice). Normalise positive counts to the number of evaluable nuclei in the same region, or report density with the measured area, and exclude folds, necrosis and poorly counterstained regions (standard IHC practice). Keep retrieval, DAB development, imaging and score thresholds consistent between sections (standard IHC practice).
How do I distinguish convincing LMO2 positivity from staining artefact?
A convincing result has a reproducible cellular pattern with nuclear staining in an appropriate population, consistent with LMO2’s nuclear annotation and HPA’s bone-marrow profile (UniProt P25791; HPA: tissue IHC profile). Treat isolated edge staining, necrotic deposits and widespread signal in the no-primary control as artefact candidates, and check endogenous peroxidase blocking when DAB appears without primary antibody (standard IHC practice). Unexpected cytoplasmic signal needs particular caution: HPA reports it in several tissues but also flags presumed off-target binding and pending external verification (HPA: tissue IHC profile and reliability). Compare staining across adjacent sections under the documented EDTA pH 8.0 retrieval before assigning biological meaning (datasheet M03502; standard IHC practice).
Boster reagents
Best LMO2 / Rhombotin-2 IHC Antibodies
M03502 has real IHC images from paraffin sections of mouse lung and a rat specimen described as a lymph node from lung tissue; no IF image is supplied (M03502 image captions; catalog IF images).
Real IHC data
Anti-LMO2 Monoclonal Antibody
Cat # M03502
M03502 is listed for IHC, with paraffin-section images from mouse lung and a rat specimen described as a lymph node from lung tissue (catalog applications; M03502 image captions). Human, mouse and rat reactivity is listed, while the supplied IHC images show mouse and rat specimens (catalog reactivity; M03502 image captions).
Which to pick: For tissue IHC, choose M03502, a rabbit monoclonal shown at 1:50 on paraffin sections; the fixative is unreported (catalog host and clone; M03502 image captions). No IF/ICC pick is supported because M03502 has no listed IF/ICC application or IF image (catalog applications; catalog IF images). For cross-species work, M03502 lists human, mouse and rat reactivity, with IHC images for mouse and rat specimens (catalog reactivity; M03502 image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P25791 (RBTN2_HUMAN, Rhombotin-2).
- Human Protein Atlas. LMO2 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. LMO2 subcellular location (ICC-IF): Mainly localized to the nucleoplasm..
- Human Protein Atlas. LMO2 antibody validation summary (4 antibodies).
- T-Cell Lymphoblastic Lymphoma Arising in the Setting of Myeloid/Lymphoid Neoplasms with Eosinophilia: LMO2 Immunohistochemistry as a Potentially Useful Diagnostic Marker. Cancers 2021 — PMC8234657.
- LMO2 expression is frequent in T-lymphoblastic leukemia and correlates with survival, regardless of T-cell stage. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 2022 — PMC9427670.
- The transcription factor LMO2 is a robust marker of vascular endothelium and vascular neoplasms and selected other entities. American journal of clinical pathology 2009 — PMC4305438.
- Round-robin testing for LMO2 and MYC as immunohistochemical markers to screen MYC rearrangements in aggressive large B-cell lymphoma. Virchows Archiv : an international journal of pathology 2024 — PMC11329383.
- PubMed PMID:1923511 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:9129143 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for LMO2 (Rhombotin-2). Expected localisation Nuclear in marrow; cytoplasmic in several tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet M03502). Validated IHC antibody: M03502 at 1:50 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded lymph node of rat lung tissue; fixative not specified (datasheet M03502); 2. Fixation: Image fixative and duration unreported (datasheet M03502); 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 M03502); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M03502); 8. Primary antibody: Rabbit monoclonal (clone ACOA-12) anti-LMO2, 1:50 (datasheet M03502); 9. Primary incubation: Overnight at 4 °C (datasheet M03502). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Presumed off-target staining; verification pending (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope differences unreported (UniProt); Regulation: Expression regulators are not specified (UniProt). Evidence: UniProt P25791, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm., Datasheet M03502, PMC8234657, PMC9427670. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/LMO2/immunohistochemistry-ihc-guide.