P2.49. A RAPID IMMUNOFLUORESCENCE METHOD COMPATIBLE WITH FUNCTIONAL ASSAYS IN ACUTE AND ORGANOTYPIC SLICES
Aleksandra Duchnowska1,2, Aleksandra Owczarek1, Aleksandra Skweres1,2, Martyna Podgajna1, Julia Łukasiewicz1, Michał Węgrzynowicz1
1 Laboratory of Molecular Basis of Neurodegeneration, Mossakowski Medical Research Institute, Polish Academy of Sciences, 5 Pawińskiego St., 02-106, Warsaw, Poland
2 Doctoral School of Translational Medicine, Centre of Postgraduate Medical Education, 99/103 Marymoncka St., 01-813 Warsaw, Poland
INTRODUCTION: Acute and organotypic slices are a powerful tool for short- and long-term experimental manipulations. They support a broad range of imaging-based functional assays using genetically-encoded or chemical fluorescent probes to study cell injury, calcium fluxes, membrane transport and more. Post-assay immunofluorescence can reveal spatially resolved protein expression, providing complementary insight into additional cellular processes and tissue structure. However, properties of acute and organotypic slices differ from fixed brain, making regular immunofluorescence protocols incompatible.
AIM(S): We aimed to develop a rapid immunofluorescence protocol for both slice types, enabling detection of selected proteins following imaging-based functional assays for direct co-analysis or post-imaging comparisons.
METHOD(S): Acute hippocampal slices were obtained from adult mice. Organotypic slices were cultured from hippocampi of rat or mice pups using standard protocols. Slices were subjected to experimental treatment and functional assays, followed by testing of fixation and immunostaining conditions to find parameters effective for both types with minimal processing time.
RESULTS: The method allows precise immunofluorescent labeling of brain regions (e.g., hippocampal CA fields) and cell types (e.g., astrocytes, neurons, CA2 pyramidal neurons ) as well as detection of condition-dependent changes in expression of proteins involved in defined cell pathways (e.g. mitochondrial calcium uptake or caspase activation). The procedure takes ~4-5 hours after overnight fixation and is compatible with prior functional assays (e.g. cell labeling, neuronal injury or polyamine uptake).
CONCLUSIONS: This method combines speed, structure preservation and compatibility with functional assays, supporting multi-dimensional analyses in basic and translational neurobiology.
FINANCIAL SUPPORT: National Science Centre, Poland: 2023/49/N/NZ4/02660, 2023/07/X/NZ4/00420, 2023/51/B/NZ4/02605.