Systematic development of immunohistochemistry protocol for large cryosections-specific to non-perfused fetal brain.

BACKGROUND: Immunohistochemistry (IHC) is an important technique in understanding the expression of neurochemical molecules in the developing human brain. Despite its routine application in the research and clinical setup, the IHC protocol specific for soft fragile fetal brains that are fixed using the non-perfusion method is still limited in studying the whole brain. NEW METHOD: This study shows that the IHC protocols, using a chromogenic detection system, used in animals and adult humans are not optimal in the fetal brains. We have optimized key steps from Antigen retrieval (AR) to chromogen visualization for formalin-fixed whole-brain cryosections (20 µm) mounted on glass slides. RESULTS: We show the results from six validated, commonly used antibodies to study the fetal brain. We achieved optimal antigen retrieval with 0.1 M Boric Acid, pH 9.0 at 70°C for 20 minutes. We also present the optimal incubation duration and temperature for protein blocking and the primary antibody that results in specific antigen labeling with minimal tissue damage. COMPARISON WITH EXISTING METHODS: The IHC protocol commonly used for adult human and animal brains results in significant tissue damage in the fetal brains with little or suboptimal antigen expression. Our new method with important modifications including the temperature, duration, and choice of the alkaline buffer for AR addresses these pitfalls and provides high-quality results. CONCLUSION: The optimized IHC protocol for the developing human brain (13-22 GW) provides a high-quality, repeatable, and reliable method for studying chemoarchitecture in neurotypical and pathological conditions across different gestational ages.

Enhanced mechanical properties of hBN-ZrO2 composites and their biological activities on Drosophila melanogaster: synthesis and characterization.

In this study, six compositions in the system [x(h-BN)-(100 - x)ZrO2] (10 ≤ x ≤ 90) were synthesized by a bottom up approach, i.e., the solid-state reaction technique. XRD results showed the formation of a novel and main phase of zirconium oxynitrate ZrO(NO3)2 and SEM exhibited mixed morphology of layered and stacked h-BN nanosheets with ZrO2 grains. The composite sample 10 wt% h-BN + 90 wt% ZrO2 (10B90Z) showed outstanding mechanical properties for different parameters, i.e., density (3.12 g cm-3), Young's modulus (10.10 GPa), toughness (2.56 MJ m-3), and maximum mechanical strength (227.33 MPa). The current study further checked the in vivo toxicity of composite 10B90Z and composite 90B10Z using Drosophila melanogaster. The composite 10B90Z showed less cytotoxicity in this model, while the composite 90B10Z showed higher toxicity in terms of organ development as well as internal damage of the gut mostly at the lower concentrations of 1, 10, and 25 µg mL-1. Altogether, the current study proposes the composite 10B90Z as an ideal compound for applications in biomedical research. This composite 10B90Z displays remarkable mechanical and biological performances, due to which we recommend this composition for various biomedical applications.