RORB Antibody

What is RORB and why is it an important research target?

RORB (RAR-related orphan receptor B) is a transcription factor belonging to the nuclear receptor family. It functions as a ligand-inducible transcription factor that regulates various aspects of mammalian physiology . RORB plays a critical role in cortical layer development, particularly in layer 4 (L4) neuronal specification. Research has demonstrated that RORB is involved in a mutually repressive interaction with Brn1/2, which is essential for proper L2/3 and L4 specification in the neocortex . This interaction makes RORB a valuable target for neurodevelopmental research, particularly studies focused on cortical layering and neuronal subtype specification.

What applications can RORB antibody be used for in research?

RORB antibodies have been validated for multiple research applications:

It is important to note that optimal dilutions may be sample-dependent, and researchers should titrate the antibody in each testing system to obtain optimal results .

How should I select the appropriate RORB antibody for my specific research question?

When selecting a RORB antibody, consider the following criteria:

• Target specificity: Determine if you need to detect all RORB variants or a specific isoform. Identify whether distinguishing between different domains (e.g., DNA-binding domain vs. ligand-binding domain) is critical for your research question .

• Species reactivity: Confirm that the antibody has been validated in your species of interest. Current RORB antibodies typically show reactivity with human, mouse, and rat samples .

• Application compatibility: Ensure the antibody has been validated for your intended application (WB, IP, IHC, IF, ELISA). Some antibodies perform well in certain applications but not in others .

• Clone type: Consider whether a monoclonal or polyclonal antibody is more appropriate for your research. Monoclonal antibodies offer high specificity for a single epitope, while polyclonal antibodies recognize multiple epitopes and may provide stronger signals .

• Validation data: Review the published literature and manufacturer's validation data to assess antibody performance. Look for evidence of specificity, such as knockout/knockdown controls .

What are the recommended protocols for antigen retrieval when using RORB antibody in IHC?

For effective immunohistochemical detection of RORB in brain tissue, the following antigen retrieval methods are recommended:

• Primary recommendation: Tris-EDTA (TE) buffer at pH 9.0. This appears to provide optimal antigen retrieval for RORB epitopes in fixed brain tissue .

• Alternative method: Citrate buffer at pH 6.0 can also be used if the primary method does not yield satisfactory results .

The specific protocol should include:

• Deparaffinization and rehydration of tissue sections

• Immersion in preheated retrieval buffer

• Heating at 95-100°C for 15-20 minutes

• Gradual cooling to room temperature

• Thorough washing before proceeding with blocking and primary antibody incubation

Optimization may be required for specific tissue preparation methods and fixation conditions.

How should RORB antibody be stored and handled to maintain its activity?

To preserve antibody activity and specificity:

• Storage temperature: Store at -20°C. RORB antibody remains stable for one year after shipment when properly stored .

• Buffer composition: The antibody is typically provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Aliquoting: While the manufacturer notes that aliquoting is unnecessary for -20°C storage, it is generally good practice to divide the antibody into small aliquots to avoid repeated freeze-thaw cycles, particularly for applications requiring high sensitivity .

• Working dilutions: Prepare fresh working dilutions on the day of use whenever possible. Store working dilutions at 4°C for short-term use (1-2 days).

• Handling precautions: Avoid contamination by using clean pipette tips and sterile tubes. Some preparations contain BSA (0.1%) as a stabilizer, which should be noted for applications where this might interfere .

What controls should be included when using RORB antibody in experiments?

Robust experimental design requires appropriate controls:

• Positive tissue control: Include samples known to express RORB, such as mouse or rat brain tissue, particularly regions with documented RORB expression like specific cortical layers .

• Negative tissue control: Use tissues known not to express RORB, or regions within the same sample that do not express RORB.

• Technical negative controls:

  • Primary antibody omission control: Apply only secondary antibody to detect non-specific binding

  • Isotype control: Use a non-specific antibody of the same isotype (Rabbit IgG for many RORB antibodies)

• Knockdown/knockout validation: For definitive specificity confirmation, include samples where RORB expression has been reduced by RNA interference or genetic knockout .

• Competing peptide control: Pre-incubating the antibody with the immunizing peptide should abolish specific staining in a concentration-dependent manner.

How can RORB antibody be used to study the mutual repression between RORB and Brn1/2 in cortical development?

The mutually repressive interaction between RORB and Brn1/2 can be investigated using several antibody-based approaches:

• Co-immunostaining analysis: Use antibodies against both RORB and Brn1/2 to demonstrate their mutually exclusive expression patterns in developing cortical layers. This provides spatial information about their relationship during development .

• ChIP (Chromatin Immunoprecipitation): RORB antibody can be used to identify direct binding of RORB to regulatory regions of the Brn2 gene. Research has identified two putative ROR binding sites approximately 8 kb upstream of the Brn2 transcription start site .

• Luciferase reporter assays: Combined with ChIP data, RORB antibody can help validate binding sites by correlating RORB binding with transcriptional repression of reporter constructs containing the identified binding sites .

• Manipulation experiments: RORB antibody can be used to validate knockdown or overexpression efficiency in experiments designed to manipulate RORB levels and observe effects on Brn1/2 expression. For instance, immunohistochemistry for RORB confirms the efficiency of shRNA-mediated knockdown .

• Developmental time course: Immunostaining with RORB antibody at different developmental stages can reveal the temporal dynamics of RORB expression in relation to Brn1/2 during cortical layer formation .

What are the technical challenges in detecting RORB in specific neuronal subtypes?

Detecting RORB in specific neuronal populations presents several technical challenges:

• Cell-type specificity: RORB expression is largely restricted to layer 4 cortical neurons, making detection in other neuronal subtypes challenging due to low expression levels .

• Temporal expression dynamics: RORB expression changes during development, requiring careful consideration of developmental timing when designing experiments .

• Background signal in neural tissue: Brain tissue often exhibits high background, particularly in immunofluorescence applications, requiring careful optimization of blocking and washing steps.

• Epitope accessibility: The nuclear localization of RORB may require enhanced permeabilization protocols, particularly for in vitro cultured neurons or thick tissue sections.

• Distinguishing between direct and indirect effects: When studying RORB's role in neuronal specification, it can be challenging to distinguish between direct effects of RORB and secondary effects caused by altered expression of downstream targets. This requires complementary molecular approaches beyond antibody-based detection .

• Co-detection with other markers: For comprehensive neuronal classification, RORB often needs to be co-detected with other markers like Cux1 and Tbr1, requiring careful antibody pairing to avoid species cross-reactivity issues .

How can RORB antibody be combined with other molecular tools to investigate neuronal positioning and projection patterns?

RORB antibody can be integrated with multiple complementary approaches to study neuronal positioning and axonal projections:

• Retrograde tracing with immunostaining: Combine retrograde tracers (like FluoroGold) with RORB immunostaining to correlate RORB expression with specific projection patterns. This approach has revealed that RORB expression reduces callosal projections in layer 2/3 neurons, while RORB knockdown increases callosal projections in E14.0-born neurons .

• In utero electroporation with immunocytochemistry: Introduce genetic constructs (e.g., for RORB overexpression or knockdown) through in utero electroporation, followed by RORB antibody staining to confirm manipulation and analyze effects on positioning and morphology .

• Multi-channel imaging: Combine RORB antibody with antibodies against cytoskeletal markers or cell adhesion molecules to investigate how RORB affects the cellular machinery involved in neuronal positioning.

• Tissue clearing with immunolabeling: Use modern tissue clearing techniques (CLARITY, iDISCO, etc.) combined with RORB immunostaining to visualize the three-dimensional organization of RORB-expressing neurons and their projections throughout the brain.

• Single-cell transcriptomics validation: Use RORB antibody to validate protein expression in subpopulations identified through single-cell RNA sequencing approaches, linking transcriptomic profiles to spatial organization.

What are common sources of false positive and false negative results when using RORB antibody?

Understanding potential artifacts is crucial for accurate data interpretation:

Sources of false positives:

• Cross-reactivity: Antibodies may recognize proteins with similar epitopes to RORB, particularly other nuclear receptors. This risk is higher with polyclonal antibodies .

• Non-specific binding: High antibody concentrations can lead to binding to non-target proteins, especially in protein-rich tissues like brain .

• Inadequate blocking: Insufficient blocking can result in non-specific binding of the primary or secondary antibody to endogenous Fc receptors or highly charged cellular components.

• Endogenous peroxidase/phosphatase activity: In enzyme-based detection systems, endogenous enzyme activity can generate signal independent of antibody binding.

Sources of false negatives:

• Epitope masking: Fixation can alter protein conformations or create protein-protein crosslinks that obscure the epitope recognized by the antibody. This is particularly relevant for nuclear proteins like RORB .

• Insufficient antigen retrieval: RORB detection in IHC requires specific antigen retrieval conditions (TE buffer pH 9.0 or citrate buffer pH 6.0) .

• Antibody degradation: Improper storage or handling can reduce antibody activity over time. Always store according to manufacturer recommendations .

• Suboptimal incubation conditions: Incorrect temperature, duration, or antibody concentration can reduce detection sensitivity.

How can I resolve discrepancies between RORB antibody results and mRNA expression data?

When antibody-based protein detection does not align with mRNA expression data:

• Post-transcriptional regulation: Differences between mRNA and protein levels may reflect genuine biological phenomena such as miRNA regulation, protein stability differences, or post-translational modifications. These are not technical artifacts but important biological insights.

• Epitope modification: Post-translational modifications might mask the epitope recognized by the antibody without affecting protein expression. Consider using multiple antibodies targeting different RORB epitopes.

• Spatial-temporal differences: RORB protein might be expressed in different cellular compartments or at different times compared to its mRNA. Perform careful time-course experiments and subcellular localization studies.

• Antibody validation: Re-validate antibody specificity using positive and negative controls. Consider knockdown/knockout controls if available .

• Technical approach validation: For mRNA detection, validate primer specificity and efficiency. For protein detection, try alternative applications (e.g., if WB shows discrepancy with IHC, validate the antibody in both applications).

• Quantification methods: Ensure that quantification methods for both mRNA and protein are appropriate and standardized. Different normalization controls might contribute to apparent discrepancies.

What are the best practices for quantifying RORB expression in immunohistochemistry and immunofluorescence experiments?

For reliable quantification of RORB expression:

• Consistent tissue processing: All experimental and control samples must undergo identical fixation, sectioning, antigen retrieval, and staining procedures.

• Standardized image acquisition:

  • Use identical exposure settings for all samples

  • Avoid saturated pixels that compromise quantification

  • Include calibration standards when possible

  • Capture multiple representative fields per sample

• Appropriate controls for normalization:

  • Include nuclear counterstain (e.g., DAPI) for cell counting

  • Use neuronal markers (e.g., NeuN) to normalize to total neuronal population

  • Include internal reference regions with stable RORB expression

• Quantification methods:

  • For binary expression (positive/negative): Establish clear thresholds for positive cells based on background levels in negative controls

  • For expression levels: Use mean fluorescence intensity with background subtraction

  • For distribution analysis: Bin cells by expression level or use cumulative distribution functions

• Double-blind analysis: When possible, have image quantification performed by researchers blinded to experimental conditions.

• Statistical considerations:

  • Use appropriate statistical tests based on data distribution

  • Account for nested data structures (multiple measurements per animal)

  • Report both effect sizes and statistical significance

How can RORB antibody be used to investigate the role of RORB in neurological disorders?

RORB antibody can advance understanding of neurological disorders through several approaches:

• Expression analysis in disease models: Compare RORB expression patterns in animal models of neurodevelopmental disorders (e.g., autism, schizophrenia) to identify alterations in cortical layer development and neuronal specification .

• Human post-mortem studies: Analyze RORB expression in post-mortem brain tissues from patients with neurological disorders to identify disease-associated changes in expression or localization.

• Functional studies with disease-associated variants: Use RORB antibody to validate expression and localization of RORB variants associated with neurological disorders in cellular models.

• Therapeutic target validation: For disorders where RORB signaling might be therapeutically targeted, use antibody-based assays to confirm target engagement and pathway modulation by candidate compounds.

• Circuit-level analysis: Combine RORB immunostaining with circuit tracing techniques to investigate how alterations in RORB-expressing neurons affect neural circuit formation and function in disease models.

What emerging technologies might enhance the utility of RORB antibody in neurodevelopmental research?

Several cutting-edge approaches can extend RORB antibody applications:

• Spatial transcriptomics validation: Use RORB antibody to validate and calibrate spatial transcriptomics data, bridging the gap between transcriptomic profiles and spatial organization of neuronal subtypes.

• Super-resolution microscopy: Apply techniques like STORM, PALM, or SIM with RORB antibody to study the nanoscale organization of RORB within the nucleus and its colocalization with other transcription factors and chromatin regions.

• In vivo antibody-based sensors: Develop genetically encoded sensors based on RORB antibody fragments to monitor RORB expression or localization dynamics in living cells.

• Mass cytometry (CyTOF): Incorporate metal-conjugated RORB antibodies into CyTOF panels to simultaneously analyze RORB expression alongside dozens of other markers in single cells.

• Organoid models: Apply RORB antibody to cerebral organoids to study the development and organization of RORB-expressing neurons in three-dimensional human neural tissue models.

• Multiplexed imaging: Use cyclic immunofluorescence or multiplexed ion beam imaging with RORB antibody to simultaneously visualize dozens of markers in the same tissue section, providing comprehensive cellular phenotyping.