romo1 Antibody

What is the most reliable method for detecting Romo1 expression in tissue samples?

Immunohistochemistry (IHC) staining is the most widely employed technique for evaluating Romo1 protein expression in tissue samples. The standard protocol involves:

• Preparing 4-micron-thick sections of paraffin-embedded tumor tissues

• Performing antigen retrieval by heating slides at 98°C for 20 minutes and cooling for 10 minutes in Epitope Retrieval Solution 1 (0.01 M citrate buffer, pH 6.0)

• Blocking endogenous peroxidase activity

• Incubating with Romo1 monoclonal antibody (typically at 1:200 dilution)

• Counterstaining with hematoxylin after development with 3,3'-diaminobenzidine chromogen solution

For quantification, histologic scoring (H-score) is recommended, calculated by multiplying staining intensities (0-3) by the percentage of cells with each intensity level (possible range: 0-300) .

How should Romo1 expression be evaluated in immune cells?

For immune cells, particularly macrophages which show notable Romo1 expression, a combination of approaches is recommended:

• Western blotting: For protein level detection in isolated immune cells (T cells, B cells, macrophages, dendritic cells)

• RT-qPCR: For mRNA expression analysis

• Immunofluorescence: Using appropriate antibodies (Romo1 + lineage markers such as CD11b for monocytes/macrophages)

Research shows that Romo1 is expressed at relatively higher levels in macrophages compared to other immune cells, making proper validation crucial for accurate analysis .

What controls should be used when validating Romo1 antibodies?

Proper controls for Romo1 antibody validation include:

• Positive control: Human colon adenocarcinoma tissues have been established as reliable positive controls for Romo1 staining

• Negative control: Exclusion of the primary antibody in parallel sections

• Knockdown validation: Using shRNA-mediated Romo1 knockdown cells to confirm antibody specificity

• Tissue comparison: Including both tumor and adjacent normal tissues to assess differential expression patterns

Comparison with Romo1 mRNA expression data can provide additional validation of antibody specificity and performance .

How can Romo1 antibodies be used to investigate the role of Romo1 in macrophage polarization?

Romo1 plays a significant role in macrophage polarization, particularly promoting M2 polarization through the mTORC1 signaling pathway. To investigate this:

• Double immunofluorescence staining: Use Romo1 antibody alongside M1 markers (iNOS) and M2 markers (CD206) to assess polarization state

• Flow cytometry: For quantification of M1/M2 markers in Romo1-overexpressing or Romo1-knockdown macrophages

• Functional assays: Measure cytokine production (IL-10, TGF-β, TNF-α, IL-6) to assess the anti-inflammatory or pro-inflammatory status of macrophages in relation to Romo1 expression

Research findings indicate that Romo1 overexpression increases production of anti-inflammatory cytokines (IL-10, TGF-β) while decreasing pro-inflammatory cytokines (TNF-α, IL-6), suggesting its role in promoting an immunosuppressive tumor microenvironment .

What are the optimal protocols for using Romo1 antibodies in glioblastoma research models?

For glioblastoma research, which has shown significant Romo1 involvement, the following protocols are recommended:

• Tumor microenvironment analysis:

  • IHC or immunofluorescence staining of tumor sections with Romo1 and CD11b antibodies to identify tumor-associated macrophages

  • Quantification of infiltrating CD3+ T cells in relation to Romo1 expression

• Bone marrow-derived macrophage (BMDM) studies:

  • Western blotting to validate Romo1 overexpression or knockdown in BMDMs

  • DCF-DA and MitoSOX staining to measure ROS levels in relation to Romo1 expression

  • Assessment of BMDM polarization using CD206 and iNOS antibodies

• In vivo models:

  • Orthotopic injection of GL261 cells in mice with Romo1-modified bone marrow cells

  • Tumor growth measurement and survival analysis

  • Combination studies with therapies like anti-PD-1

Research has demonstrated that Romo1 inhibition in bone marrow cells significantly inhibits glioblastoma growth and prolongs survival in mouse models, highlighting its potential as an immunotherapy target .

How can Romo1 antibodies be utilized to study the relationship between Romo1 and cellular ROS production?

To investigate the relationship between Romo1 and ROS production:

Research shows that Romo1 overexpression promotes ROS accumulation and may lead to mitochondrial dysfunction in macrophages, influencing their polarization and function within the tumor microenvironment .

What are the most common technical challenges when using Romo1 antibodies?

Common technical challenges include:

• Background staining: This may occur due to:

  • Non-specific binding of the primary or secondary antibody

  • Inadequate blocking of endogenous peroxidase activity

  • Cross-reactivity with other proteins

  Solution: Optimize antibody dilutions (typically 1:200 is effective), use appropriate blocking reagents, and include proper negative controls .

• Variable staining intensity: This challenge is particularly relevant when comparing different patient samples or experimental conditions.

  Solution: Use standardized histologic scoring (H-score) methods that account for both staining intensity and percentage of positive cells .

• Detection sensitivity: Particularly in cells with lower Romo1 expression levels.

  Solution: Consider signal amplification methods or more sensitive detection systems for low-expressing samples.

How should researchers analyze conflicting Romo1 expression data between protein and mRNA levels?

When encountering discrepancies between protein and mRNA expression data:

• Validate antibody specificity using knockdown or knockout controls

• Consider post-transcriptional regulation mechanisms that might affect protein levels independently of mRNA

• Analyze samples using multiple methodologies (western blot, IHC, RT-qPCR)

• Assess the potential influence of the tumor microenvironment on protein stability

Recent studies have demonstrated that evaluating Romo1 gene expression may be more reliable than protein expression in some contexts, as mRNA overexpression has been correlated with unfavorable prognosis in gastric cancer and bladder cancer .

What are the best approaches for studying Romo1's role in drug resistance mechanisms?

To investigate Romo1's involvement in drug resistance:

• Paired sample analysis:

  • Compare Romo1 expression in pre-treatment and post-resistance tumor samples

  • Use IHC or western blotting with Romo1 antibodies

• Functional studies:

  • Generate Romo1-overexpressing and knockdown cell lines

  • Assess sensitivity to therapeutic agents (e.g., platinum compounds, EGFR-TKIs)

  • Measure ROS levels in relation to drug sensitivity

• Combination therapy models:

  • Test Romo1 inhibition in combination with standard therapies

  • Assess potential synergistic effects, particularly with immune checkpoint inhibitors like anti-PD-1

Research has demonstrated that high Romo1 expression is associated with poor response to platinum-based chemotherapy in advanced NSCLC and with shorter progression-free survival in EGFR-mutated lung adenocarcinoma treated with TKIs .

How can Romo1 antibodies be applied in studying the relationship between Romo1 and immune checkpoint inhibitor efficacy?

For investigating Romo1's role in immune checkpoint inhibitor efficacy:

• Tumor microenvironment analysis:

  • Multi-color immunofluorescence with Romo1, PD-1/PD-L1, and immune cell markers

  • Assessment of tumor-infiltrating lymphocytes in relation to Romo1 expression

• Combinatorial therapy studies:

  • In vivo models combining Romo1 inhibition with PD-1 blockade

  • Analysis of survival outcomes and immunological changes

• Patient sample correlation:

  • Retrospective analysis of Romo1 expression in patients treated with immune checkpoint inhibitors

  • Correlation with response rates and survival outcomes

Research has shown that combination of Romo1 inhibition with anti-PD-1 immunotherapy significantly improved survival outcomes in glioblastoma mouse models, suggesting potential synergistic effects .

What methodologies are recommended for investigating Romo1's function as a mitochondrial cation channel?

To study Romo1's channel activity:

• Electrophysiological approaches:

  • Patch-clamp techniques to measure ion conductance

  • Assessment of channel activity in response to specific inhibitors or modulators

• Mitochondrial function analysis:

  • Measurement of mitochondrial membrane potential (Δψm)

  • Analysis of mitochondrial calcium flux

  • Assessment of mitochondrial ROS production in relation to channel activity

• Structural studies:

  • Structural modeling based on experimental data

  • Analysis of the amphipathic helical transmembrane domain necessary for pore-forming activity

Research has identified that Romo1 forms a nonselective cation channel with viroporin-like characteristics, and its activity is specifically inhibited by Fe²⁺ ions, which are essential for ROS metabolism. The inhibitory concentration (IC₅₀) for Fe²⁺ (0.4 μM) falls within the cytosolic free iron concentration range (0.2-1.5 μM) .

What approaches should be used to study the relationship between Romo1 and cellular metabolism in cancer?

To investigate Romo1's role in cellular metabolism:

• Metabolic profiling:

  • Extracellular acidification rate (ECAR) measurement to assess glycolysis

  • Oxygen consumption rate (OCR) analysis for oxidative phosphorylation

  • Comparison between Romo1-overexpressing and control cells under different metabolic stresses

• Glucose metabolism assessment:

  • 2-NBDG uptake assays to measure glucose uptake

  • Analysis of glucose transporter expression (Glut1, Glut3) in relation to Romo1 levels

  • ATP production measurement

• Signaling pathway analysis:

  • Investigation of mTORC1 pathway activation

  • Assessment of metabolic enzyme expression and activity

Research findings demonstrate that Romo1 overexpression promotes glycolysis while inhibiting oxidative phosphorylation in macrophages, suggesting its role in cellular metabolic reprogramming that may influence tumor progression .

What are the most promising approaches for developing Romo1-targeted therapies?

Based on current research, several approaches show promise:

• Direct Romo1 inhibition:

  • Development of small molecule inhibitors targeting Romo1's channel function

  • RNAi-based approaches for specific Romo1 knockdown

• Combination therapy strategies:

  • Pairing Romo1 inhibition with immune checkpoint blockade

  • Combining with conventional therapies (chemotherapy, radiation) to overcome resistance

• Cell-specific targeting:

  • Approaches to specifically target Romo1 in tumor-associated macrophages

  • Methods to modulate macrophage polarization by targeting Romo1-dependent pathways

Research has demonstrated that inhibition of Romo1 in bone marrow cells, combined with anti-PD-1 immunotherapy, significantly improved survival outcomes in glioblastoma mouse models, highlighting the potential of this approach .

How should researchers design experiments to investigate microRNA regulation of Romo1 expression?

For studying microRNA regulation of Romo1:

• Bioinformatic prediction:

  • Identify potential miRNA binding sites in Romo1 mRNA

  • Focus on miRNAs differentially expressed in relevant cancer types

• Validation experiments:

  • Luciferase reporter assays with wild-type and mutated Romo1 3'UTR

  • miRNA mimic and inhibitor transfection followed by Romo1 expression analysis

  • Correlation analysis between miRNA and Romo1 expression in patient samples

• Functional studies:

  • Assess the impact of miRNA modulation on Romo1-dependent phenotypes

  • Investigate potential therapeutic applications of miRNA-mediated Romo1 regulation

Recent research has identified that LINC00319, a long non-coding RNA, functions as a sponge for miR-4492, which directly targets Romo1 expression. This miR-4492/Romo1 axis has been shown to regulate proliferation, migration, and tumor invasion in bladder cancer cells .

What methodological approaches are recommended for studying Romo1's role in different tissue and organ systems?

For investigating Romo1's function across different tissues:

• Tissue-specific knockout models:

  • Generation of conditional Romo1 knockout mice using tissue-specific Cre recombinase systems

  • Analysis of phenotypic changes and disease susceptibility

• Comparative expression analysis:

  • Systematic comparison of Romo1 expression across different tissues

  • Correlation with tissue-specific metabolic and functional parameters

• Disease model studies:

  • Investigation of Romo1's role in tissue-specific pathologies

  • Assessment of potential tissue-specific therapeutic applications