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LDB3 / LIM domain-binding protein 3 · IHC design guide
Design Immunohistochemistry for LDB3
Plan LDB3 paraffin-section IHC using heart and skeletal muscle as positive tissues, with cytoplasmic staining expected in their muscle cells (HPA tissue IHC). This guide covers consistent fixation, chromogenic detection, the catalog antibody’s 1:50–1:200 IHC dilution (datasheet A04633), and isoform-aware interpretation (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
LDB3 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 in heart and skeletal muscle (HPA tissue IHC) | |
| Staining pattern | Cardiomyocyte cytoplasm high; skeletal myocyte cytoplasm medium (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, heat-mediated (datasheet A04633) | |
| Positive control | Heart muscle+2 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 | Staining may detect proteins from more than one gene (HPA tissue IHC) | |
| Regulation | Heart high; skeletal muscle medium (HPA tissue IHC) | |
| Isoform / epitope | Seven isoforms; check epitope coverage; no extracellular domain (UniProt) |
Section 1
Recommended LDB3 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is paired with two published LDB3 IHC protocols (PMC9822979; PMC9636405).
| Sample | Paraffin-embedded mouse stomach tissue; fixative not specified (datasheet A04633) |
| Fixation | Image fixative and duration unreported (datasheet A04633); 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 6.0 (datasheet A04633); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-LDB3, 1:50-1:200 (datasheet A04633) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | LDB3-positive staining in cardiomyocytes of heart muscle (HPA tissue IHC: High). HPA tissue profile: Selective cytoplasmic expression in heart and skeletal muscle. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated citrate retrieval at pH 6.0 (datasheet: A04633); PMC9822979 reports a 20 min citrate retrieval.
Section 2
What Is the Expected LDB3 Staining Pattern?
LDB3 is a cytoplasmic, Z-line-associated protein with no transmembrane segment (UniProt O75112). In paraffin-section IHC, expect staining chiefly in cardiomyocytes and skeletal myocytes, with possible staining in late spermatids (HPA tissue IHC: High, Medium, and Medium, respectively). HPA rates the tissue pattern Supported, while cautioning that the antibody targets proteins from more than one gene (HPA tissue IHC).
| Strong cytoplasmic staining in cardiomyocytes, with less intense staining in skeletal myocytes. | This fits the reported High cardiomyocyte and Medium skeletal-myocyte signals (HPA tissue IHC). A striated pattern would be compatible with LDB3 at sarcomeric Z-lines (UniProt O75112), but HPA's tissue summary establishes cytoplasmic expression, not a required striation pattern (HPA tissue IHC). |
| Medium staining in elongated or late spermatids, or weak staining in placental trophoblastic cells. | These are reported outside the principal muscle pattern: Medium in late spermatids and Low in trophoblastic cells (HPA tissue IHC). Judge them by their stated cell types and levels; neither finding alone overrides the stronger heart and skeletal-muscle pattern (HPA tissue IHC). |
| Predominantly nuclear staining in cardiomyocytes or skeletal myocytes. | A nuclear-dominant IHC result conflicts with the expected cytoplasmic muscle pattern (HPA tissue IHC; UniProt O75112). Investigate background and antibody specificity before scoring it as LDB3. An uncertain nucleoplasmic ICC-IF location does not establish nuclear muscle IHC staining (HPA subcellular). |
| Strong staining in adipocytes or other cells listed as Not detected. | This disagrees with the listed HPA cell-level observations, including adipocytes in adipose tissue (HPA tissue IHC). Check whether staining follows tissue structures or detection background; cross-reactivity or endogenous chromogen activity are possible general IHC explanations, not demonstrated causes in this specimen. |
| Broad, even colour across cells and surrounding tissue, without a clear cytoplasmic muscle pattern. | Treat this as background until controls show otherwise. The selective cytoplasmic heart and skeletal-muscle profile provides the comparison (HPA tissue IHC). General IHC causes include incomplete blocking, concentrated antibody, or residual detection activity; appearance alone cannot identify which occurred. |
💡Expected LDB3 appearanceCall the result positive when cardiomyocytes show predominantly cytoplasmic staining at the reported High level, with skeletal myocytes typically Medium (HPA tissue IHC); nuclear-dominant muscle staining or strong signal in HPA Not detected cell types needs control-based review.
| Tissue and cell selection | Heart muscle cardiomyocytes provide the clearest reported positive reference (High); skeletal-muscle myocytes are Medium (HPA tissue IHC). Adipose-tissue adipocytes are listed as Not detected (HPA tissue IHC). Compare named cell populations, because the tissue-level label alone does not identify the stained cells. |
| Intracellular location | LDB3 has no transmembrane segment and is assigned to cytoplasm, cytoskeleton, myofibrils and sarcomeric Z-lines (UniProt O75112). Interpret muscle IHC against the cytoplasmic HPA profile; do not require a resolved Z-line pattern in every paraffin section (HPA tissue IHC). |
| Antibody evidence and specificity | The tissue-IHC reliability is Supported, with an explicit caution that staining may target proteins from more than one gene (HPA tissue IHC). HPA048955 is IHC Supported but ICC Uncertain (HPA antibodies). These ratings support pattern comparison; they do not prove that every stained structure is LDB3. |
| Isoform coverage | UniProt lists 7 LDB3 isoforms (UniProt O75112). The supplied record gives no antibody epitope or isoform coverage, so a difference between specimens cannot be assigned to an isoform from this evidence. Confirm the catalog antibody's epitope information before making an isoform-specific claim. |
| Detection chemistry | For chromogenic IHC, endogenous enzyme activity or nonspecific reagent binding can produce colour independently of target recognition (standard IHC practice). Use matched detection controls when signal appears outside the expected cell population; HPA's staining levels do not identify the chemistry behind a particular specimen's background. |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in cardiomyocytes of a heart-muscle section. | This misses the reported High reference population (HPA tissue IHC). The image alone cannot distinguish a detection failure from unsuitable antibody conditions. | Check section integrity and assay controls, then review antigen retrieval and catalog-antibody dilution as general paraffin-IHC variables. Change one condition at a time; no LDB3-specific retrieval setting or dilution is supplied. |
| Heart-muscle signal is present, but skeletal myocytes look faint. | The reported comparison is High cardiomyocytes versus Medium skeletal myocytes (HPA tissue IHC), so lower skeletal intensity may be expected. | Score each named cell type against its own reference level and confirm the cytoplasmic location (HPA tissue IHC). Avoid calling skeletal muscle negative solely because it is weaker than heart. |
| Nuclear colour dominates an otherwise positive muscle section. | The expected muscle pattern is cytoplasmic (HPA tissue IHC; UniProt O75112). Nuclear colour could reflect background or nonspecific staining; its cause is unresolved. | Compare a matched negative control and review the antibody and detection steps. Record nuclear-dominant colour separately until the cytoplasmic muscle signal can be assessed. |
| Strong colour appears in adipocytes or many unrelated cell populations. | Adipocytes are reported Not detected in adipose tissue (HPA tissue IHC). HPA also cautions that the tissue antibody may target proteins from more than one gene (HPA tissue IHC). | Inspect cell identity and negative controls; review blocking and endogenous enzyme controls as standard chromogenic-IHC checks. Do not assign the extra signal to LDB3 solely from colour. |
| Diffuse colour obscures the boundary between positive cells and surrounding tissue. | The expected profile is selective cytoplasmic staining in heart and skeletal muscle (HPA tissue IHC). Diffuse colour may arise from general staining or detection background. | Compare reagent controls, then review blocking, washing and antibody concentration as general IHC steps. Keep interpretation tied to identifiable cardiomyocytes or myocytes and their cytoplasm. |
| IF/ICC Q: Should focal-adhesion or nucleoplasmic signal count as confirmed LDB3? | HPA lists cytosol and focal adhesions as main ICC-IF locations and nucleoplasm as additional, all uncertain; it warns that the antibodies target proteins from multiple genes (HPA subcellular). HPA048955 is ICC Uncertain (HPA antibodies). | Treat those IF/ICC locations as provisional and use the separate IF/ICC guide for that application. They do not redefine the expected cytoplasmic muscle result in paraffin IHC (HPA tissue IHC). |
Experiment evidence
IHC & IF images showing LDB3 staining
A04633 · IHCImmunohistochemistry of paraffin-embedded mouse stomach using LDB3 Rabbit pAb at dilution of 1:200 .Perform high pressure antigen retrieval
A04633 · IFImmunofluorescence analysis of human placenta cells using LDB3 antibody at dilution of 1:100. Blue: DAPI for nuclear staining.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000122367-LDB3/tissue/cerebral+cortex#imid_14104094
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000122367-LDB3/tissue/colon#imid_14099276
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000122367-LDB3/tissue/liver#imid_14099269
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000122367-LDB3/tissue/kidney#imid_14099278
HPA · ICC-IF Rh30Human Protein Atlas immunofluorescence, Rh30, antibody HPA048955; green = LDB3, blue = nucleus, red = microtubules.
HPA · ICC-IF Rh30Human Protein Atlas immunofluorescence, Rh30, antibody HPA048955; green = LDB3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for LDB3 IHC & IF
🧪Run heart muscle first: cardiomyocytes should stain (HPA: High in cardiomyocytes). Use adipose tissue as a negative comparison (HPA: Not detected in adipocytes); on the heart slide, compare cardiomyocytes with surrounding noncardiomyocyte areas as a background reference, without assuming those cells are target-negative (HPA: cardiomyocyte-specific row only).
Positive control tissue: Heart muscle (Cardiomyocytes, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show LDB3 in Rh30, SK-MEL-30, U2OS, with annotated localisation: Focal adhesion sites (uncertain), Cytosol (uncertain) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species and immunoglobulin class (standard IHC practice). Confirm specificity with LDB3 knockout tissue or a validated immunizing-peptide block if available (standard IHC practice); assess endogenous peroxidase background in heart sections before interpreting chromogenic staining (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the fixative in the selected A04633 paraffin-section caption is unreported (selected-SKU caption: fixative not stated). That caption uses high-pressure retrieval in 10 mM citrate, pH 6.0, at 1:200 in mouse stomach, so retrieval dependence in heart remains unestablished (selected-SKU caption: mouse stomach IHC). There is no supplied matched evidence that frozen sections or IF are easier; in heart, distinguish specific cardiomyocyte staining from endogenous peroxidase background (HPA: High in cardiomyocytes; standard IHC practice).
HPA tissue IHC evidence for LDB3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — High consistency between antibody staining and RNA expression data. Caution, targets protein from more than one gene.). 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 |
|---|---|---|---|---|
| Heart muscle | Cardiomyocytes | High | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | Medium | Protein (IHC) | HPA → |
| Testis | Elongated or late spermatids | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | Endocrine cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced LDB3 IHC Tips
Use compartment, tissue controls and matched processing conditions to troubleshoot chromogenic LDB3 staining in paraffin sections.
How should I adjust retrieval when LDB3 staining is weak or uneven?
Start with heat-mediated retrieval in 10 mM citrate, pH 6.0, using high pressure for paraffin sections (datasheet A04633). The pictured mouse stomach section was stained at 1:200, but its retrieval duration was not reported (datasheet A04633). If staining is weak, vary heating duration in small steps while keeping section thickness, cooling and antibody dilution constant (standard IHC practice). Compare each condition with a heart muscle or skeletal muscle control and check that muscle morphology remains intact (HPA: cytoplasmic staining in heart and skeletal muscle; standard IHC practice). Excessive heating can damage morphology and make apparent gains in staining difficult to interpret (standard IHC practice).
Can fixation explain weak LDB3 staining in paraffin sections?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not report a fixative, and no LDB3 fixation comparison is supplied (datasheet A04633). Record the fixative, fixation interval, tissue size and processing schedule for every specimen before comparing staining intensity (standard IHC practice). Run differently processed specimens alongside a consistently processed muscle control, keeping retrieval and detection conditions identical (HPA: cytoplasmic staining in heart and skeletal muscle; standard IHC practice). If signal changes with processing, test matched sections across a controlled fixation series rather than assigning the change to LDB3 biology (standard IHC practice). Preserve morphology when choosing a workable condition (standard IHC practice).
Which staining pattern should count as LDB3 localisation?
In striated muscle, look for cytoplasmic staining with a myofibrillar or Z-line pattern; LDB3 colocalizes with ACTN2 at Z lines (UniProt O75112: localisation). Heart cardiomyocytes stain highly and skeletal myocytes moderately in the tissue profile, although those antibody data carry a multiple-gene caution (HPA: tissue IHC). Inspect longitudinal muscle fibres for repeatable striations, and score them separately from diffuse cytoplasmic colour (UniProt O75112: Z-line localisation; standard IHC practice). Perinuclear cytoplasm is also described, so a perinuclear signal alone needs a matching positive control and careful cell identification (UniProt O75112: localisation; standard IHC practice). Treat isolated nuclear staining cautiously (HPA: uncertain nucleoplasmic localisation).
Why might a validated antibody miss LDB3 in my sections?
LDB3 has 7 reported isoforms, so establish which sequence the antibody recognizes before comparing specimens or declaring a negative result (UniProt O75112: isoforms; standard IHC practice). Its PDZ domain spans residues 1–84, while three LIM domains span 549–727; an unspecified epitope cannot be assigned to either region (UniProt O75112: domains). Retrieve the immunogen or mapped epitope information for the catalog antibody, then compare it with the isoform sequences under study (standard IHC practice). Phosphorylated residues include 44, 121 and 123, but the supplied evidence does not establish an effect on antibody binding (UniProt O75112: modified residues). Interpret absent staining with a known positive tissue control (HPA: heart muscle staining; standard IHC practice).
How can I check LDB3 localisation by multiplex IF?
Pair LDB3 with an ACTN2 channel to assess Z-line alignment in striated muscle, using separately acquired channels and a merged image (UniProt O75112: ACTN2 colocalisation; standard IF practice). Select spectrally separated fluorophores and place the weaker signal in a channel with low measured tissue autofluorescence (standard IF practice). LDB3 is cytoplasmic and lacks a transmembrane segment, so use a controlled permeabilisation step that permits access to its intracellular epitope (UniProt O75112: localisation and topology; standard IF practice). Titrate permeabilisation and antibody concentration on matched sections, then inspect single-label and secondary-only controls for bleed-through and background (standard IF practice). Do not transfer the paraffin-section retrieval caption to IF without testing it (datasheet A04633; standard IF practice).
How do I reduce diffuse brown staining without losing LDB3 signal?
Start with a no-primary control and a matched positive muscle section to separate detection background from expected cytoplasmic staining (HPA: heart and skeletal muscle staining; standard IHC practice). For peroxidase-based chromogenic detection, block endogenous peroxidase before applying the detection reagent and develop DAB for a controlled interval (standard IHC practice). Titrate the catalog antibody below and above the pictured 1:200 dilution, holding retrieval and development conditions fixed (datasheet A04633; standard IHC practice). Include appropriate protein blocking and washes, then compare muscle fibres with nearby structures on the same section (standard IHC practice). Diffuse colour without reproducible cellular localisation should not be scored as specific LDB3 staining (UniProt O75112: localisation; standard IHC practice).
What is a defensible way to quantify LDB3 IHC? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scoring compartment as myocyte cytoplasm before viewing experimental groups, because tissue IHC describes cytoplasmic muscle staining (HPA: tissue IHC; standard IHC practice). Report the percentage of positive myocytes and staining intensity, or calculate an H-score on a 0–300 scale using intensity categories 0–3 (standard IHC practice). Normalise counts or positive area to evaluable muscle area in mm², excluding folds, necrosis and empty space by a prespecified rule (standard IHC practice). Keep section thickness, retrieval, imaging and DAB development matched, and record the threshold used for positivity (standard IHC practice). Analyse heart and skeletal muscle separately because their reported staining levels differ (HPA: heart High; skeletal muscle Medium).
When is a brown signal convincing evidence of LDB3?
A convincing result follows the expected cytoplasmic distribution in identified muscle cells and reproduces in a concurrently processed positive control (UniProt O75112: localisation; HPA: heart and skeletal muscle; standard IHC practice). Repeated Z-line-like staining supports the assignment, while predominantly isolated nuclear colour needs caution because nucleoplasmic localisation is uncertain in the supplied cell data (UniProt O75112: Z lines; HPA: subcellular). Examine section edges, folds and necrotic regions for staining that tracks tissue damage rather than intact cells (standard IHC practice). Use no-primary and peroxidase-block controls to investigate DAB colour arising from the detection system or endogenous enzyme activity (standard IHC practice). The tissue profile has a multiple-gene antibody caution, so staining alone does not establish LDB3-specific identity (HPA: reliability description).
Boster reagents
Best LDB3 / LIM domain-binding protein 3 IHC Antibodies
A04633 has pictured LDB3 IHC in mouse stomach paraffin sections and IF in human placenta and rat heart cells (catalog image captions); its listed reactivity includes human, mouse and rat (catalog reactivity).
Real IHC data
Anti-LIM domain-binding protein 3 LDB3 Antibody
Cat # A04633
A04633 renders with mouse stomach paraffin-section IHC at 1:200 after citrate retrieval at pH 6.0 (A04633 IHC image caption). Its IF captions show human placenta cells and rat heart cells at 1:100, with DAPI nuclear staining (A04633 IF image captions).
Which to pick: Choose A04633 for tissue IHC when the pictured mouse stomach paraffin-section result is relevant; its caption reports citrate retrieval but does not report the fixative (A04633 IHC image caption). Choose A04633 for IF/ICC because both applications are listed and IF images are supplied; A04633-2 also lists IF/ICC but has no supplied IF figure (catalog applications and image captions). For work across human, mouse and rat, A04633 lists all three species and is identified as a rabbit polyclonal antibody, although its pictured IHC result is in mouse only (catalog reactivity; A04633 IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O75112 (LDB3_HUMAN, LIM domain-binding protein 3).
- Human Protein Atlas. LDB3 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. LDB3 subcellular location (ICC-IF): Mainly localized to the cytosol and focal adhesion sites. In addition localized to the nucleoplasm. Caution: Based on antibodies targeting proteins from multiple genes..
- Human Protein Atlas. LDB3 antibody validation summary (1 antibodies).
- Expression of LIM domain-binding 3 (LDB3), a striated muscle Z-band alternatively spliced PDZ-motif protein in the nervous system. Scientific reports 2023 — PMC9822979.
- Identification of novel prognostic and predictive biomarkers in salivary duct carcinoma via comprehensive molecular profiling. NPJ precision oncology 2022 — PMC9636405.
- Differences in aberrant expression and splicing of sarcomeric proteins in the myotonic dystrophies DM1 and DM2. Acta neuropathologica 2010 — PMC4199327.
- PubMed PMID:10427098 — UniProt-cited evidence.
- PubMed PMID:9734811 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for LDB3 (LIM domain-binding protein 3). Expected localisation Cytoplasmic in heart and skeletal muscle (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, heat-mediated (datasheet A04633). Validated IHC antibody: A04633 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Heart muscle (Cardiomyocytes); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded mouse stomach tissue; fixative not specified (datasheet A04633); 2. Fixation: Image fixative and duration unreported (datasheet A04633); 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 6.0 (datasheet A04633); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-LDB3, 1:50-1:200 (datasheet A04633); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Staining may detect proteins from more than one gene (HPA tissue IHC); Isoform / epitope: Seven isoforms; check epitope coverage; no extracellular domain (UniProt); Regulation: Heart high; skeletal muscle medium (HPA tissue IHC). Evidence: UniProt O75112, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the cytosol and focal adhesion sites. In…, Datasheet A04633, PMC9822979, PMC9636405. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/LDB3/immunohistochemistry-ihc-guide.