CRABP2 Antibody

Which tissue fixation protocols are most compatible with CRABP2 immunohistochemistry?

For CRABP2 immunohistochemistry, formalin fixation with paraffin embedding (FFPE) has demonstrated reliable results in multiple studies. The research protocol described used paraffin-embedded blocks of surgically resected primary PDACs processed using a BenchMark Ultra automated immunostainer . For optimal antigen retrieval in FFPE tissues, heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) is generally recommended. Tissue fixation time should be standardized (typically 12-24 hours) to ensure consistent immunoreactivity while preserving tissue morphology.

How should CRABP2 antibody positive controls be selected for different experimental applications?

Selecting appropriate positive controls for CRABP2 antibody validation is critical. Based on research findings, lung adenocarcinoma tissue samples with known CRABP2 overexpression serve as effective positive controls for immunohistochemistry and Western blotting . For cell-based assays, cell lines with documented high CRABP2 expression, such as certain LUAD cell lines, provide reliable positive controls. The mean plasma level of CRABP2 in LUAD patients was reported as 31.6587 ±13.8541 ng/mL compared to 13.9328 ± 5.5805 ng/mL in healthy controls , offering quantitative benchmarks for expression levels in different sample types.

What are the critical considerations when designing CRABP2 knockdown experiments to study its functional role?

When designing CRABP2 knockdown experiments, researchers should consider:

• Selection of appropriate knockdown technology: RNA interference technology was successfully employed in LUAD studies . CRISPR/Cas9 gene editing has also proven effective for establishing CRABP2 knockout cell lines, particularly in pancreatic cancer research .

• Validation of knockdown efficiency: Quantitative PCR and Western blotting should be used to confirm knockdown efficiency, with multiple siRNA/shRNA sequences tested to identify the most effective constructs.

• Phenotypic assessment strategy: Based on CRABP2's known functions, experiments should assess:

  • Cell cycle progression (particularly G2/M transition)

  • Apoptosis (using flow cytometry and apoptosis detection)

  • Cell proliferation (using assays such as CCK-8)

  • Changes in immune checkpoint molecule expression

• Control selection: Proper controls, including scrambled siRNA/shRNA or non-targeting gRNAs, are essential to distinguish specific effects from off-target consequences.

How should researchers design experiments to investigate the relationship between CRABP2 expression and immune checkpoint molecules?

To investigate CRABP2's relationship with immune checkpoint molecules, researchers should:

• Establish baseline correlations: Analyze datasets from resources like TCGA and GEO to establish correlation patterns between CRABP2 and immune checkpoint genes (CD274/PD-L1, PDCD1/PD-1, CTLA4, LAG3, TIGIT, HAVCR2, PDCD1LG2/PD-L2, IGSF8) .

• Design expression manipulation experiments:

  • Overexpression and knockdown/knockout of CRABP2 in relevant cell lines

  • Measure consequent changes in immune checkpoint molecule expression using qPCR and Western blotting

  • Flow cytometry to assess surface expression of checkpoint proteins

• Co-culture experiments: Design co-culture systems with immune cells to assess functional consequences of CRABP2 modulation on immune cell activation and function.

• Validate in tissue samples: Perform multiplexed immunohistochemistry or immunofluorescence to assess co-localization and expression patterns in patient samples.

Research indicates that low CRABP2 expression may enhance CD274(PD-L1), HAVCR2, and PDCD1LG2(PD-L2) expression, while high CRABP2 expression may enhance CTLA4, LAG3, PDCD1(PD-1), TIGIT, and IGSF8 expression in LUAD .

What controls are essential when using CRABP2 antibody for quantitative assessment of expression levels in patient samples?

For quantitative assessment of CRABP2 expression in patient samples, essential controls include:

• Technical controls:

  • Antibody specificity controls (peptide competition or knockout/knockdown samples)

  • Isotype controls to assess non-specific binding

  • Secondary antibody-only controls

• Sample processing controls:

  • Standardized fixation and processing protocols

  • Batch controls to normalize between processing runs

• Quantification controls:

  • Internal reference standards with known CRABP2 concentrations

  • Calibration curves for absolute quantification

• Normalization controls:

  • Housekeeping proteins for Western blotting (β-Actin was used at 1:1000 dilution in referenced studies)

  • Reference genes for qPCR

  • Matched normal tissue controls from the same patient when possible

In the LUAD plasma study, researchers established a cut-off value of 0.6551 ng/mL for CRABP2, yielding 70.98% sensitivity and 94.53% specificity with an Area Under the Curve of 0.839 (95%CI: 0.817-0.859, p<0.0001) .

How can CRABP2 antibody be utilized to investigate its role in tumor immune microenvironment regulation?

CRABP2 antibody can be employed in several advanced techniques to investigate its role in the tumor immune microenvironment:

• Multiplex immunohistochemistry/immunofluorescence:

  • Co-staining CRABP2 with immune cell markers (CD8, CD4, CD68, etc.)

  • Spatial analysis of CRABP2-expressing cells relative to immune cell infiltration

  • Correlation with immune checkpoint molecule expression

• Flow cytometry and cell sorting:

  • Isolating CRABP2-high versus CRABP2-low cells for functional studies

  • Assessing correlation with immune checkpoint molecules at single-cell level

• Single-cell RNA sequencing integration:

  • Correlating CRABP2 expression with immune cell transcriptional signatures

  • Investigating cellular heterogeneity in tumors based on CRABP2 expression

• Chromatin immunoprecipitation (ChIP) studies:

  • Investigating if CRABP2 directly or indirectly regulates immune checkpoint gene expression

Research has shown that high CRABP2 expression inhibits recruitment of immune effector cells while promoting immunosuppressive cell populations in LUAD . Specifically, CRABP2 expression correlates with B cell memory, T cell CD4+ memory (activated and resting), Tregs, T cell follicular helper, and other immune cell populations as assessed through CIBERSORT algorithm analysis .

What methodological approaches can be used to investigate the potential role of CRABP2 in regulating cholesterol metabolism?

To investigate CRABP2's role in cholesterol metabolism, consider these methodological approaches:

• Lipid raft isolation and characterization:

  • Sucrose gradient ultracentrifugation to isolate lipid rafts

  • LC-MS-MS analysis to assess cholesterol content in CRABP2-modulated cells

• Metabolic labeling experiments:

  • Use of radiolabeled or fluorescently labeled cholesterol precursors

  • Pulse-chase experiments to track cholesterol synthesis and trafficking

• Gene expression and pathway analysis:

  • RNA-seq or microarray analysis comparing CRABP2 knockout/knockdown versus control cells

  • Focus on genes involved in cholesterol biosynthesis, uptake, efflux, and metabolism

  • Validation of key targets with qPCR and Western blotting

• Protein interaction studies:

  • Co-immunoprecipitation (Co-IP) to identify CRABP2 interacting partners involved in cholesterol metabolism

  • RNA-IP to identify mRNAs associated with CRABP2 that encode cholesterol metabolism proteins

Recent findings have identified CRABP2 as a novel regulator of cholesterol metabolism, particularly in the context of pancreatic cancer drug resistance .

How can CRABP2 antibody be utilized in high-throughput screening for potential therapeutic targeting strategies?

CRABP2 antibody can facilitate high-throughput screening for therapeutic targeting through:

• Cell-based screening platforms:

  • Development of reporter cell lines with CRABP2 expression coupled to fluorescent/luminescent readouts

  • High-content screening using automated microscopy with CRABP2 antibody staining

  • Correlation of compound effects with CRABP2 localization or expression level changes

• Targeted protein degradation approaches:

  • Screening for compounds that induce CRABP2 degradation (similar to SNIPER-11 mentioned in pancreatic cancer research)

  • Western blotting with CRABP2 antibody to assess degradation efficiency

• Combinatorial drug screening:

  • Testing drug combinations with CRABP2 modulators

  • Assessing synergistic effects, particularly with chemotherapeutic agents like gemcitabine

  • Using CRABP2 antibody-based readouts to monitor expression changes

• Patient-derived organoid/xenograft screening:

  • Establishing PDX models with varying CRABP2 expression levels

  • Testing drug response correlated with CRABP2 expression

  • Immunohistochemical analysis using CRABP2 antibody

Research has demonstrated that targeting CRABP2 can overcome pancreatic cancer drug resistance, suggesting similar approaches may be valuable in other cancer types where CRABP2 is implicated .

What are the common sources of variability in CRABP2 immunohistochemistry results, and how can they be mitigated?

Common sources of variability in CRABP2 immunohistochemistry include:

• Pre-analytical variables:

  • Tissue fixation time and conditions

  • Storage duration of paraffin blocks

  • Sectioning thickness

  Mitigation: Standardize fixation protocols (12-24 hours in 10% neutral buffered formalin), use recently cut sections, and maintain consistent section thickness (4-5 μm).

• Analytical variables:

  • Antigen retrieval methods

  • Antibody concentration and incubation conditions

  • Detection systems

  Mitigation: Use automated staining platforms (like the BenchMark Ultra automated immunostainer mentioned ), optimize antigen retrieval conditions, and standardize antibody dilution (1:1000 dilution has been validated) .

• Post-analytical variables:

  • Interpretation criteria

  • Scoring systems

  • Observer variability

  Mitigation: Implement digital pathology platforms (like the ScanScope XT mentioned ), establish clear scoring criteria, and have multiple independent observers (as noted in the research: "Immunoreactivity was scored by 2 investigators independently") .

• Antibody lot-to-lot variation:

  • Different production batches may have varying specificity/sensitivity

  Mitigation: Validate each new antibody lot against known positive controls, maintain reference samples, and consider using monoclonal antibodies when possible.

How should researchers address contradictory findings regarding CRABP2 expression and function across different tumor types?

When facing contradictory findings regarding CRABP2 across tumor types:

• Context-dependent analysis:

  • Recognize that CRABP2 may have tissue-specific and tumor-specific functions

  • Analyze results in the specific context of the tissue/tumor microenvironment

  • Consider developmental lineage of the tissue in question

• Methodological standardization:

  • Compare experimental methodologies between contradictory studies

  • Standardize antibody usage, detection methods, and scoring systems

  • Ensure appropriate controls were included in all studies

• Isoform-specific analysis:

  • Determine if studies distinguished between potential CRABP2 isoforms

  • Use antibodies that specifically recognize relevant isoforms

  • Verify at the mRNA level which isoforms are being expressed

• Integrated multi-omics approach:

  • Combine protein expression data with mRNA expression, genomic, and epigenomic data

  • Use multiple large public databases (TCGA, GEO, GEPIA2, UALCAN, etc.) as was done in the LUAD study

  • Validate findings across independent cohorts

• Functional validation:

  • Perform parallel knockdown/knockout experiments across different cell types

  • Test multiple functional readouts (proliferation, apoptosis, migration, etc.)

  • Consider genetic background differences between model systems

What are the critical factors to consider when comparing CRABP2 expression data from different methodological approaches (IHC vs. Western blot vs. ELISA)?

When comparing CRABP2 expression data across different methodological platforms:

• Differential detection characteristics:

  • IHC: Semi-quantitative, preserves spatial information, detects localization

  • Western blot: Semi-quantitative, confirms molecular weight, detects protein modifications

  • ELISA: Highly quantitative, high throughput, limited spatial information

  Consideration: Use complementary techniques to validate findings, recognizing each method's strengths and limitations.

• Sample preparation differences:

  • IHC: Fixed tissues, epitope retrieval challenges

  • Western blot: Protein extraction efficiency, denaturation

  • ELISA: Native protein conformation, matrix effects

  Consideration: Standardize extraction protocols, account for extraction efficiency, and validate antibody performance in each context.

• Quantification approaches:

  • IHC: H-scores, percentage positive cells, intensity scales

  • Western blot: Densitometry, normalization to loading controls

  • ELISA: Absolute concentration, standard curves

  Consideration: Develop cross-platform normalization strategies and conversion factors when possible.

• Dynamic range and sensitivity:

  • IHC: Limited dynamic range, subjective scoring

  • Western blot: Medium dynamic range, semi-quantitative

  • ELISA: High dynamic range, highly quantitative

  Consideration: Match technique sensitivity to expected expression levels; use ELISA for low abundance detection.

• Reference standards and controls:

  • Ensure consistent use of reference materials

  • Include internal controls common across platforms

  • Consider spike-in controls for quantification

The LUAD study established plasma CRABP2 levels using quantitative methods (31.6587 ±13.8541 ng/mL in LUAD patients vs. 13.9328 ± 5.5805 ng/mL in controls) , providing a reference point for comparisons with tissue expression by other methods.

What are the most promising approaches for developing CRABP2-targeted therapeutic strategies based on current antibody research?

Based on current antibody research, promising CRABP2-targeted therapeutic approaches include:

• Targeted protein degradation:

  • Development of SNIPER (Specific and Non-genetic IAP-dependent Protein ERaser) compounds that induce selective degradation of CRABP2, similar to SNIPER-11 used in pancreatic cancer research

  • Proteolysis-targeting chimeras (PROTACs) that could target CRABP2 for ubiquitin-proteasome degradation

• Immune checkpoint modulation:

  • Combinatorial approaches targeting both CRABP2 and immune checkpoints based on the observed correlations

  • Customized immunotherapy strategies based on CRABP2 expression levels, as research suggests patients with high CRABP2 expression may respond differently to various checkpoint inhibitors

• Antibody-drug conjugates (ADCs):

  • Development of CRABP2-targeting antibodies conjugated to cytotoxic payloads

  • Selective delivery to CRABP2-overexpressing tumor cells

• Synergistic combination therapies:

  • Identification of compounds that synergize with CRABP2 inhibition

  • Combination with standard chemotherapies (gemcitabine showed synergistic effects when combined with CRABP2 targeting in PDAC)

• Biomarker-guided treatment selection:

  • Utilizing CRABP2 antibodies for patient stratification to guide therapy selection

  • Development of companion diagnostics for CRABP2-targeted therapies

How might advanced antibody engineering techniques enhance CRABP2 detection sensitivity and specificity for clinical diagnostic applications?

Advanced antibody engineering could enhance CRABP2 diagnostics through:

• Affinity maturation:

  • Directed evolution or rational design approaches to increase antibody affinity

  • Yeast or phage display technologies to select higher-affinity variants

  • Single-domain antibodies with improved tissue penetration properties

• Fragment-based approaches:

  • Development of Fab or scFv fragments with improved tissue penetration

  • Bispecific antibodies targeting CRABP2 and tumor-specific markers

  • Nanobodies with superior stability and tissue penetration

• Signal amplification strategies:

  • Polymer-based detection systems with multiple secondary antibodies

  • Quantum dot conjugation for improved signal-to-noise ratio

  • Proximity ligation assay (PLA) for detection of CRABP2 protein interactions

• Multiplexed detection platforms:

  • Development of antibody panels for simultaneous detection of CRABP2 and related biomarkers

  • Mass cytometry (CyTOF) applications for highly multiplexed single-cell analysis

  • Spatial transcriptomics integration with antibody-based detection

• Automation and standardization:

  • Development of automated platforms for consistent CRABP2 detection

  • Digital pathology solutions with AI-assisted interpretation

  • Standardized reference materials for cross-laboratory comparison

The current diagnostic accuracy of CRABP2 in LUAD (sensitivity: 70.98%, specificity: 94.53%, AUC: 0.839) could potentially be improved through these advanced engineering approaches.

What are the key methodological considerations for investigating CRABP2's role in modulating response to immunotherapy across different cancer types?

Key methodological considerations for investigating CRABP2's immunotherapy modulation role include:

• Comprehensive baseline characterization:

  • Extensive profiling of CRABP2 expression across cancer types using standardized antibody-based methods

  • Correlation with immune checkpoint molecule expression (CD274/PD-L1, PDCD1/PD-1, CTLA4, etc.)

  • Multi-parameter flow cytometry to evaluate immune cell populations in relation to CRABP2 expression

• Preclinical model development:

  • Generation of isogenic cell lines with CRABP2 knockout/overexpression

  • Development of syngeneic mouse models with modulated CRABP2 expression

  • Patient-derived xenograft models stratified by CRABP2 expression levels

• Functional immune assays:

  • T-cell killing assays comparing CRABP2-high versus CRABP2-low targets

  • Cytokine profiling in the tumor microenvironment

  • Immune cell migration and infiltration assays

• Clinical correlation studies:

  • Retrospective analysis of CRABP2 expression in responders versus non-responders to immunotherapy

  • Prospective collection of pre- and post-treatment biopsies to assess CRABP2 dynamics

  • Liquid biopsy approaches to monitor CRABP2 levels during immunotherapy

• Mechanistic investigation approaches:

  • ChIP-seq to identify CRABP2 genomic binding sites related to immune function

  • ATAC-seq to assess chromatin accessibility changes in relation to CRABP2 expression

  • Protein interaction studies to identify CRABP2 binding partners in immune regulation

Research indicates CRABP2 expression could predict response to specific immune checkpoint inhibitors: patients with high CRABP2 expression may have suboptimal response to inhibitors targeting CD274/HAVCR2/PDCD1LG2 but better response to inhibitors targeting CTLA4/LAG3/PDCD1/TIGIT/IGSF8 .