NPFFR2 Antibody, FITC conjugated

What is NPFFR2 and what molecular pathways does it regulate?

NPFFR2 (neuropeptide FF receptor 2), also known as GPR74, NPFF2, and NPGPR, belongs to the G-protein coupled receptor 1 family. It functions as a receptor for NPAF (A-18-F-amide) and NPFF (F-8-F-amide) neuropeptides, which are known as morphine-modulating peptides . NPFFR2 mediates its action by association with G proteins that activate a phosphatidylinositol-calcium second messenger system . Recent research has demonstrated that NPFFR2 activation stimulates the hypothalamic-pituitary-adrenal (HPA) axis and can trigger anxiety-like behaviors, indicating its significant role in stress responses and behavioral regulation .

What tissues show positive NPFFR2 expression?

Based on immunohistochemistry studies, NPFFR2 expression has been reliably detected in:

NPFFR2 has also been detected in specific brain regions, particularly in hypothalamic nuclei where it plays roles in neuroendocrine regulation .

How do I select between NPFFR2 and NPFFR1 antibodies for my research?

Selection should be based on:

• Receptor subtype specificity: NPFFR2 has distinct physiological roles compared to NPFFR1, with NPFFR2 being more strongly implicated in HPA axis regulation and stress responses .

• Cross-reactivity profile: Some antagonists like BIBP3226 show differential affinity (NPFFR1 > NPFFR2), which is important when designing pharmacological experiments .

• Experimental model: Different tissues exhibit varying expression patterns of NPFFR1 versus NPFFR2.

When studying anxiety-related behaviors or HPA axis function, NPFFR2-specific antibodies are typically more appropriate as direct hypothalamic NPFFR2 stimulation has been shown to trigger anxiety-like behaviors in animal models .

What is the optimal protocol for immunohistochemistry with NPFFR2 antibodies?

Based on validated experimental approaches:

• Tissue preparation: Fix tissues in 4% paraformaldehyde and embed in paraffin or prepare frozen sections.

• Antigen retrieval: Use TE buffer pH 9.0 (preferred) or alternatively citrate buffer pH 6.0 .

• Blocking: 5-10% normal serum (species matched to secondary antibody) in PBS with 0.3% Triton X-100.

• Primary antibody: Dilute NPFFR2 antibody between 1:20-1:200 depending on application specificity . For FITC-conjugated versions, initial testing at 1:50 is recommended with subsequent optimization.

• Incubation: Overnight at 4°C or 2 hours at room temperature.

• Visualization: For unconjugated primary antibodies, use appropriate secondary antibodies. For FITC-conjugated antibodies, wash thoroughly and proceed directly to mounting.

• Mounting: Use antifade mounting medium to preserve fluorescence.

Note that each specific system may require antibody titration to obtain optimal results .

How should NPFFR2 antibodies be validated for specificity?

Multi-step validation is essential:

• Preabsorption controls: Incubate the antibody with excess target peptide (10μM NPFF) before application to tissue. Loss of signal confirms specificity .

• Cross-reactivity testing: Test against related peptides like NPY to rule out non-specific binding. For example, verification that FMRFa antibodies do not cross-react with NPY is critical when studying NPFF systems .

• Knockout/knockdown validation: Where available, tissues from NPFFR2 knockout or knockdown models provide gold standard controls.

• Multiple antibody comparison: Use antibodies targeting different epitopes of NPFFR2 to confirm staining patterns.

• Multiple detection methods: Confirm protein expression using complementary techniques (Western blot, RNA in situ hybridization).

What controls are essential when using FITC-conjugated NPFFR2 antibodies?

For fluorescence-based detection:

• Autofluorescence control: Examine unstained tissue sections to identify natural autofluorescence.

• Isotype control: Use FITC-conjugated isotype-matched immunoglobulin (Rabbit IgG for rabbit polyclonal antibodies) at equivalent concentration .

• Secondary antibody control: Include samples with secondary antibody only (when using indirect methods).

• Cross-channel bleed-through control: Especially important in multiplex experiments with additional fluorophores.

• Signal specificity control: Pre-incubate antibody with NPFFR2 peptide to verify signal elimination.

How can NPFFR2 antibodies be used to study HPA axis activation?

NPFFR2 antibodies can be employed to investigate the relationship between NPFFR2 expression and HPA axis activation:

• Colocalization studies: Use FITC-conjugated NPFFR2 antibodies with other markers of HPA axis activation (e.g., CRF, ACTH, glucocorticoid receptors).

• Activation tracking: Combine with c-Fos immunostaining to determine which NPFFR2-expressing neurons are activated during stress responses.

• Receptor trafficking: Monitor NPFFR2 localization changes following administration of agonists like AC-263093 or dNPA that have been shown to dose-dependently increase serum corticosterone levels .

• Quantitative analysis: Measure NPFFR2 expression levels before and after stress challenges or pharmacological treatments.

Research has demonstrated that NPFFR2 agonists (AC-263093, CFMHC, dNPA) dose-dependently increase serum corticosterone levels, with effects that can be inhibited by CRF antagonists, confirming the role of NPFFR2 in HPA axis regulation .

How do NPFFR2 isoforms affect antibody binding and experimental results?

NPFFR2 exists in multiple isoforms (isoform1, isoform2, and isoform3) , which can impact experimental outcomes:

• Antibody epitope selection: The 19505-1-AP antibody recognizes all three known isoforms of NPFFR2 , but researchers should verify epitope specificity when working with other antibodies.

• Isoform-specific expression: Different tissues may express varying ratios of NPFFR2 isoforms.

• Functional differences: Isoforms may couple differently to downstream signaling pathways.

• Experimental design implications: When studying specific functions, consider using isoform-specific antibodies if available.

When interpreting results, particularly in comparative studies across tissues, consider that observed differences might reflect isoform distribution rather than total NPFFR2 expression.

What are the experimental considerations for studying NPFFR2-mediated signaling pathways?

NPFFR2 activates specific signaling cascades that should be considered when designing experiments:

Research has shown that NPFF and RFRP-3 (GnIH) signaling through their receptors involves pertussis toxin-sensitive G proteins and cesium-sensitive potassium channels, with differential effects on downstream pathways .

What are common technical issues with FITC-conjugated antibodies in NPFFR2 detection?

Several technical challenges may arise:

• Photobleaching: FITC is susceptible to photobleaching. Use antifade mounting media and minimize exposure to light during sample handling.

• Autofluorescence: Particularly problematic in tissues with high lipofuscin content. Consider using Sudan Black B (0.1-0.3%) treatment to reduce autofluorescence.

• pH sensitivity: FITC fluorescence is optimal at alkaline pH. Ensure buffers maintain appropriate pH.

• Fixation artifacts: Overfixation can mask epitopes. Optimize fixation protocols for specific tissues.

• Signal-to-noise ratio: Background may be higher with direct conjugates compared to amplified detection systems. Optimize antibody concentration and blocking conditions.

How can researchers distinguish between specific and non-specific binding?

Multiple approaches should be employed:

• Absorption tests: Pre-incubate antibody with excess antigenic peptide (NPFF or NPFFR2-specific peptides) to confirm signal elimination .

• Cross-reactivity assessment: Test antibody against related proteins (e.g., NPY) to ensure specificity .

• Concentration gradients: Titrate antibody to determine optimal concentration for specific signal.

• Multiple antibody validation: Compare results with antibodies targeting different NPFFR2 epitopes.

• Tissue-specific controls: Include tissues known to be negative for NPFFR2 expression.

Research has demonstrated that careful validation is essential, as some antibodies targeting RFamide peptides show cross-reactivity. For example, some FMRFa antibodies can cross-react with NPY, necessitating specific controls .

What statistical approaches are recommended for analyzing NPFFR2 expression data?

Statistical analysis should be tailored to the experimental design:

• For dose-response studies: One-way ANOVA followed by Newman-Keuls post hoc tests for independent factors, as employed in studies of NPFFR2 agonist-induced corticosterone elevation .

• For time-course experiments: Repeated measures two-way ANOVA followed by Bonferroni post hoc tests .

• For comparing expression levels: Unpaired Student's t-test for independent factors .

• For multiple condition comparisons: One-way ANOVA with appropriate post hoc tests (Bonferroni, Tukey, etc.) .

• Sample size considerations: Based on published studies, typical experimental designs should include 4-11 samples per group to achieve adequate statistical power .

How might FITC-conjugated NPFFR2 antibodies advance research on stress and anxiety disorders?

FITC-conjugated NPFFR2 antibodies could enable several novel research approaches:

• Live cell imaging: Monitor receptor trafficking and internalization in real-time following ligand binding or stress stimuli.

• FACS analysis: Isolate NPFFR2-positive cell populations from complex tissues for transcriptomic or proteomic profiling.

• Optogenetic integration: Combine with optogenetic approaches to correlate receptor expression with functional outcomes.

• Brain region mapping: Create detailed maps of NPFFR2 expression across brain regions involved in stress responses.

Current research has established that NPFFR2 activation stimulates the HPA axis and induces anxiety-like behaviors , providing a foundation for using FITC-conjugated antibodies to further elucidate these mechanisms.

What emerging techniques might benefit from NPFFR2 antibody applications?

Several cutting-edge methodologies could utilize NPFFR2 antibodies:

• Spatial transcriptomics: Correlate NPFFR2 protein expression with local transcriptional profiles.

• Super-resolution microscopy: Examine subcellular localization of NPFFR2 with nanometer precision.

• Expansion microscopy: Physically expand tissues to reveal fine details of NPFFR2 distribution.

• Multiplexed imaging: Simultaneously visualize multiple components of NPFFR2 signaling networks.

• Single-cell proteomics: Analyze NPFFR2 expression variability within seemingly homogeneous cell populations.