Multiplexed immunofluorescence and RNA-FISH

This protocol describes a multiplexed immunofluorescence and RNA-FISH method to detect Atp13a4 mRNA in astrocytes of the visual cortex at postnatal stages. Brain sections from P7, P14, and P21 Aldh1L1-EGFP mice are immunostained with anti-GFP antibodies to label astrocytes and hybridized with custom Atp13a4 probes. Amplification is performed using hairpins specific to the probes and initiator-labeled antibodies. Sections are imaged using high-resolution confocal microscopy, and Atp13a4 puncta are quantified within GFP-positive astrocyte regions using a FIJI-based custom pipeline. This approach enables spatially resolved analysis of Atp13a4 expression during postnatal development.