D eclipse c1si

All slices were imaged using a confocal microscope at 20x objective (Nikon D-eclipse C1si; Nikon Instruments, Melville, NY). Laser intensity and gain were held constant for each label across all slices. To obtain a broader field of view without losing resolution, each complete image contained 20 individual 20x images stitched together using Photoshop (Adobe Systems Inc., San Jose, CA). To control for naturally occurring variance in cell type density between cortical layers, complete images were cropped into individual layers according to Kandel et al. (2000 ). For some slices, the implanted devices extended past the cortex and into the white matter. These images were cropped at the end of layer six and the white matter was not analyzed.

Immunofluorescence staining was performed as described previously.¹⁷ Briefly, A549 cells were seeded on glass slides in 12‐well plates and incubated as indicated for 24 hours. The slides were washed three times with PBS, fixed in 4% paraformaldehyde for 10 minutes and then permeabilized with 0.1% Triton X‐100 for 20 minutes. Whereafter, the cells were blocked with 3% BSA at room temperature for 1 hour and subsequently incubated with primary antibody (vimentin, 1:100; p‐Erk1/2, 1:100) at 4°C overnight. After incubation, slices were washed three times with PBS and incubated with corresponding secondary antibodies (1:200; Beyotime Institute of Biotechnology, Haimen, China). The nuclei were stained with DAPI (Beyotime Institute of Biotechnology) for 5 minutes. Samples were washed three times with PBS and mounted in antifade mounting medium, and fluorescence was detected using a confocal laser scanning microscope (Nikon D‐Eclipse C1si; Nikon Corporation, Tokyo, Japan) or a fluorescence microscope (EVOSTM Auto 2; Invitrogen, WA, USA).

Immunofluorescence was carried out as previously described^{60 (link)}. PCa cells were cultured in chamber slides (Fisher Scientific, Hampton, NH), washed and fixed in 4% paraformaldehyde. After another series of washing, cells were permeabilized and blocked with 2% BSA in TBST buffer. Cells were incubated overnight at 4 °C with primary antibodies as indicated. Next, cells were incubated with Alexa Fluor® 488 secondary antibody, then stained with 4′ 6′-diamindino-2-phenylindole (DAPI) and mounting medium (Vector Laboratories, Burlingame, CA). Images were acquired under Nikon D-ECLIPSE C1si spectral laser-scanning confocal (Nikon Instruments, Melville, NY).