A Hemisphere-Separated Protocol for Morphological Observation and Combined RNA-Protein Extraction in Murine Brain Regions

In daily neuroscience research, morphological staining and nucleic acid/protein molecular assays typically require separate experimental animals, which increases animal use and is inconsistent with the 3Rs animal welfare principle. This protocol establishes a tissue allocation workflow for murine brain tissue that enables paired morphological and molecular analyses from a single animal. After transcardial perfusion with 0.9% saline, the brain is bisected into two hemispheres. One hemisphere is fixed in 4% paraformaldehyde for immunohistochemistry and immunofluorescence; sections stored in a sucrose-ethylene glycol-polyvinylpyrrolidone (PVP) solution maintain reliable staining quality for up to seven years. The contralateral hemisphere is cryopreserved at -80 °C for combined RNA and protein extraction, with yields sufficient for downstream qPCR and Western Blot. Tested on 9-10 mg entorhinal cortex punches, this method produces lower RNA and protein yields than separate extraction of RNA or protein individually, yet the recovered biomolecules remain adequate for molecular detection. This workflow lowers animal consumption and removes inter-individual differences between paired morphological and molecular analyses. It provides a feasible tissue preparation method for multidimensional profiling of discrete murine brain subregions.