Optimization of Freezing Method to Facilitate Cryosectioning of Large Brain Tissues

Cellular-level studies are essential for a comprehensive understanding of the brain tissue's intricate structure and function. While high-throughput, high-resolution histology pipelines are available for the detailed analysis of small animal brains like mice and marmosets, establishing a comparable pipeline for large brains like those of goats or humans remains challenging. Cryosectioning, known for its minimal shrinkage and preservation of histochemical activities in the brain tissue, has been standardized for small brains or 5–10 mm thick sections of large brain tissues. We have developed a standardized protocol for freezing large brain sections up to 40–50 mm thick with minimal artifacts. The protocol integrates brain master patterns, custom-designed molds, an isopentane and dry ice bath, and a continuous temperature monitoring system to ensure high-quality histology. The freezing rate, a critical factor in artifact prevention, was determined to be 27.3 °C/min for 3.2 min using an isopentane and dry ice bath for a mouse brain. Achieving similar high freezing rates in large brain tissues necessitates designing a technically complex and expensive freezing system. Hence, optimizing the key contributing factors, such as the design of the molds, the tissue orientation during freezing, the extent of the embedding medium surrounding the tissue, and the isopentane level, is crucial for enabling rapid freezing of large brain tissues. Experimental characterization and optimization were conducted using an agarose block resembling a goat brain's dimensions, serving as a surrogate for large brain tissues. Results showed that the agarose block froze at a rate of 2–3 °C/min, ideal for artifact-free freezing, when using a base mold with inner volume 125% than that of the agarose block, orienting the brain tissue to align its lowest dimension along the width of the base mold and ensuring an isopentane level covering at least 80%-90% of the frozen block's height. Application of this technique to freeze a goat brain yielded nearly 2800 sections without freezing artifacts, even in the deepest regions where freezing is expected to be slower.