A ONE-DAY POST-ASSAY IMMUNOFLUORESCENCE METHOD FOR INTEGRATION OF ANATOMICALLY RESOLVED PROTEIN EXPRESSION WITH NMDA-INDUCED EXCITOTOXICITY AND MCU-DEPENDENT CA²⁺ FLUXES IN HIPPOCAMPAL SLICES

Acute and organotypic hippocampal slices enable live imaging-based functional assays to study excitotoxic injury and intracellular Ca²⁺ dynamics, that can be integrated with post-assay immunofluorescence to obtain essential molecular and spatial information. However, slice-specific properties and prior exposure to NMDA and fluorescent probes limit the applicability of standard staining protocols developed for fixed tissue.
The aim of this study was to develop a rapid immunofluorescence protocol compatible with acute and organotypic hippocampal slices, allowing reliable detection of selected proteins following functional imaging assays and enabling direct post-assay co-analysis.
Acute hippocampal slices were prepared from adult mice and organotypic slices were cultured from neonatal rat or mouse hippocampi. Following experimental treatments, including NMDA-induced excitotoxic stimulation, slices were subjected to functional assays and subsequently processed using systematically optimized fixation and immunostaining conditions to minimize processing time while preserving tissue integrity and fluorescent signals.
The optimized protocol enables precise immunofluorescent labelling of hippocampal subregions (including CA1 and CA2) and major cell types, as well as detection of condition-dependent changes in proteins associated with defined cellular pathways, such as mitochondrial Ca²⁺ uptake and apoptotic signalling. The procedure requires approximately 4-5 hours following overnight fixation and remains fully compatible with prior live assays, including cell injury and uptake-based measurements.
This rapid immunofluorescence method combines time efficiency, structural preservation, and compatibility with functional imaging, supporting integrated analyses of excitotoxic injury and region-specific signalling mechanisms in hippocampal slice models.
This work was supported by the National Science Centre, Poland, through grants no 2023/51/B/NZ4/02605, 2023/49/N/NZ4/02660 and 2023/07/X/NZ4/00420.