Development of the Contrasting Fluorescence Immunostaining Technique for 3D Imaging of Astrocyte Ultramorphology

Abstract

Objective: Modern neurobiology focuses on understanding and preventing neurodegeneration by detecting cellular changes early. Changes in astrocyte ultramorphology may indicate early neurodegeneration, associated with decreased synaptic plasticity and astrocyte aging. While high-resolution techniques like EM and SPM can study these structures, they can’t use fluorescence microscopy for complete 3D analysis. We suggest using fluorescent dye-conjugated nanoantibodies for astrocyte labeling, allowing detailed ultramorphological research with electron, optical, and scanning probe microscopy, plus optical probe nanotomography. Methods: Hippocampi from male C57BL/6 mice were stained using commercial or GFAP-specific VHH-E9 nanoantibodies. The VHH-E9 antibodies were created by synthesizing a nucleotide sequence with added histidines and cysteine, then cloning it into the pET22 plasmid at XbaI and BamHI sites. Escherichia coli Rosetta-gami (DE3) cells were transformed to produce the pET22-VHHE9 strain. The nanoantibodies were produced via autoinduction and conjugated with a fluorophore. Astrocyte morphology was analyzed using the Sholl test with FiJi software. Results and Discussion: Traditional immunoglobulins can cause visualization inaccuracies due to the distance between the label and target protein. Nanoantibody conjugates, being smaller, reduce these errors. Immunostainings with both types showed similar efficiency, as Sholl profiles from different samples aligned within error margins. This supports the effectiveness of the sample preparation method and VHH-E9 nanoantibodies. It also suggests potential for developing similar procedures with fluorescent contrast agents, like semiconductor nanocrystals. Conclusions: To study astrocyte changes in early neurodegenerative stages, we propose combining high-resolution microscopy, optical microspectroscopy, and 3D ultrastructure restoration. Our method, OPNT, uses fluorescence microscopy for localization data, and SPM/UMT for ultrastructure. This involves creating UMT sections for simultaneous OM/SPM analysis and reconstructing them into a 3D array. We efficiently reconstruct 3D astrocyte ultramorphology and developed an immunostaining procedure using camel VHH-E9 nanoantibodies specific to GFAP, highlighting its efficiency and potential with fluorescent semiconductor nanocrystals.