RNAscope in situ hybridization combined with immunohistochemistry

Mice were anesthetized with ketamine/xylazine and perfused with ice-cold saline followed by 4% paraformaldehyde (PFA) 7 days after sham or FPI. Dissected brains were post-fixed in 4% PFA at 4 °C overnight, then cryoprotected in 30% sucrose solution until sinking. Brains were embedded in optimal cutting temperature (OCT) compound by the University of Iowa Central Microscopy Research Facility, and 10-μm coronal sections were prepared.

Frozen sections from the injury epicenter were dried at 40 °C for 30 min and RNAscope for Clec7a (ACD, 532061) and Serpina3n (ACD, 430191) was performed per manufacturer’s instructions. After RNAscope procedure, sections were placed in blocking/extraction solution (0.5% Triton X-100 and 10% goat serum in 1× PBS) for 1 h at room temperature. After blocking, tissues were incubated overnight at 4 °C in rabbit anti-GFAP (1:100; Abcam AB16997) or rabbit anti-Iba1 (1:200; Wako Chemicals 019-17941) primary antibody diluted in blocking/extraction solution. Alexa Fluor-568 or 647-conjugated goat anti-rabbit secondary antibody (Life Technologies) was used at a 1:1000 dilution for 1 h at room temperature. Fluorescently stained tissue slices were imaged using a Zeiss 710 confocal microscope. Confocal images from cortex, corpus callosum, hippocampus, and thalamus were used. ImageJ quantification of GFAP+, IBA1+, GFAP+serpina3n+, and IBA1+clec7A+ cells was performed. Positive cell numbers were counted and divided by area, and data was reported as cells per mm².