🧫 Experiment Protocol
Falsificationproposed
SUMMARY
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- Compare brain penetration in FcRn+/+ vs FcRn-/- mice with engineered vs native antibodies
- Test whether pH-modified variants retain microglia
## Background and Rationale
This falsification experiment investigates the role of the neonatal Fc receptor (FcRn) in antibody-mediated therapeutics for neuroinflammation by comparing brain penetration and therapeutic efficacy of engineered versus native antibodies in FcRn-competent and FcRn-deficient mice. The study addresses critical questions
METHODOLOGY NOTES
**Phase 1: Animal Preparation and Genotyping (Days 1-7)**
• Obtain 48 C57BL/6J mice (24 FcRn+/+ and 24 FcRn-/-) aged 8-12 weeks
• Confirm genotypes via PCR analysis of tail biopsies
• Acclimate animals for 7 days in controlled environment (12h light/dark, 22±2°C)
• Fast animals 12h before antibody administration
**Phase 2: Antibody Preparation and Characterization (Days 5-8)**
• Prepare four antibody variants: native IgG1, pH-modified IgG1 (His-substituted Fc), native anti-CD11b, pH-modified anti-CD11b
• Confirm pH-dependent FcRn binding via surface plasmon resonance (pH 6.0 vs 7.4)
• Label antibodies with fluorescent dyes (Alexa Fluor 647) for real-time tracking
• Validate antibody integrity and binding capacity via flow cytometry
**Phase 3: Real-Time Transcytosis Imaging Setup (Days 8-9)**
• Install cranial windows in subset of mice (n=6 per genotype) under isoflurane anesthesia
• Allow 48h recovery period with analgesics (buprenorphine 0.1mg/kg)
• Calibrate two-photon microscopy s