Charged glass slides

At 1 week, rats implanted with microwires were transcardially perfused with PBS, 4% paraformaldehyde, and 20% sucrose. Following decapitation, the skulls were exposed and placed in 4% paraformaldehyde overnight at 4°C and then 30% sucrose overnight at 4°C. The brains were then extracted and stored in 30% sucrose at 4°C overnight or until the brains sunk to the bottom of the container. Brains were frozen at −20°C and cryosectioned transversely onto charged glass slides (VWR, PA, USA). Slides were thawed to room temperature and washed with PBS. The slides were incubated at room temperature in blocking solution (0.4% Triton-X, 4% goat serum in PBS) for 1 h. The following primary antibodies were used: rabbit anti-GFAP (1:1,000, DAKO, CA, USA), mouse IgG1 anti-NeuN (1:500, Millipore, CA, USA), and mouse anti-CD68 (1:500, Millipore, CA, USA). Primary antibodies were diluted in blocking solution and incubated overnight at 4°C. Slides were then washed in PBS and washing solution (0.4% Triton-X in PBS). The appropriate secondary antibody was applied for 1 h at room temperature, followed by DAPI for 15 min. Slides were washed again in PBS and washing solution, dried, and coverslipped with Fluoromount-G (Southern Biotech, AL, USA). Stained slides were imaged at 10× on a Zeiss Axiovert 200 M (Carl Zeiss, NY, USA).