Advances in imaging and analysis of 4 fluorescent components through the rat cortical column

Immunofluorescent staining coupled with axial optical sectioning allows for assessment of native three-dimensional structure of brain tissue. Typical challenges of analyzing network structure include limitations driven by magnification/field of view, spatial resolution, tissue thickness, staining quality of dense cell types, data quantifiability and the quantity of simultaneous staining targets.

This manuscript demonstrates many methodological advancements. Software-aided alignment of the cortical slice and stereotaxic atlas maximizes ROI-identification accuracy. Tissue compression during antigen retrieval enhances epitope availability without damaging tissue. A thorough factorial experiment focusing on Smi-311 staining highlights the enhancements in image quality from our extended staining protocol. Mosaic scanning techniques and subsequent four-channel alignment ensures high data quality.

Cortical column datasets [800μm x 3000μm x 70μm] utilizing sequential optical sectioning were successfully generated from three rats. Each rat provided three coronal sections in each of two regions, M1 and S1BF, from which data cubes were generated per hemisphere, totaling 36 high-magnification four-color datasets.

Typical confocal assessments of brain tissue do not utilize such thick tissue slices nor collect entire cortical columns from the cortical surface to the grey/white interface at a resolution that can map fine filamentous processes. The simultaneous collection of our four specific structural markers - neuronal, astrocytic, vascular and nuclear - is novel and the quantitative optimization of staining protocols through a factorial design rare.

Building upon this preliminary success in protocol development, future work will encompass volumetric modeling and quantitative analysis of regional network architecture.