780 confocal microscope

Mice were anesthetized using isoflurane, rapidly decapitated, and brains were rapidly extracted. Immediately after removal, the brains were placed on a square of aluminum foil on dry ice to freeze. Brains were then placed in a − 80°C freezer for no more than 1 week before slicing. Slices of the BNST (12 μm) were made on a Leica CM3050S cryostat (Germany) and placed directly on coverslips. FISH was performed using the Affymetrix ViewRNA 2-Plex Tissue Assay Kit with custom probes for 5-HT_1A and Cre designed by Affymetrix (Santa Clara, CA). Slides were coverslipped with Southern Biotech DAPI Fluoromount-G (Birmingham, AL). 3 × 5 tiled z stack (15 optical sections comprising 14 μ m total) images of the entire 12 μ m slice were obtained on a Zeiss 780 confocal microscope for assessment of 5-HT_1A and Cre expression. All images were preprocessed with stitching and maximum intensity projection. An image of the BNST from 3 mice in each condition was hand counted for each study using the cell counter plugin in FIJI (ImageJ).

PC-3M cells were cultured in RPMI 1640 medium (Thermofisher) with 10% FBS (Invitrogen) and 100 units/mL of penicillin and streptomycin (Thermofisher) and trypsinized at 70% confluence. Cells (100 μL/well) were seeded at density of 2 × 10⁵ cells/mL in glass-bottom 96 well plate (Corning, cat# 4586) overnight at 37 °C, 5% CO₂. To evaluate nucleolar disassembly, media was replaced with 100 μL RMPI media containing compound or 1% DMSO and incubated 24 hr at 37 °C, 5% CO₂. Nucleolar staining was performed following protocol recommended in Nucleolar-ID^® Green Detection Kit (Cat #: ENZ-51009-500). Hoechst 33342 (Thermofisher # H3570) was used for staining nucleus. Images were captured using Zeiss 780 confocal microscope. Three separate images for each treatment conditions were analyzed using Image J software. The number of automatically counted bright objects (green fluorescent objects) was normalized to the number of Hoechst-stained nuclei. For relative comparison, the fluorescence ratio for vehicle-treated cells was set to 100.

To detect CSPGs and glial cell activation, optic nerve, spinal cord or retinal sections from at least three different animals for each antibody and condition were stained with antibodies as follows: slides were incubated for 1 h in blocking solution (PBS containing 3% goat serum and 0.2% Triton X-100 followed by overnight incubation at 4°C in blocking solution containing diluted primary antibodies. For detection of core proteins (neurocan, aggrecan and versican), a pretreatment step in which slides were treated with chondroitinase ABC overnight at 37°C was required. Following chondroitinase ABC digestion, slides were washed twice with PBS, and then followed previously described blocking and primary antibody treatment. Next, slides were washed three times for 5 min (PBS), incubated for 2 h in blocking solution containing diluted secondary antibodies, washed, and mounted using glass coverslips with Fluoromount medium (Sigma). Imaging was performed using a Zeiss 780 confocal microscope. For fluorescence localization comparison, image capture settings were held constant, and samples from within each experimental group were imaged at the same time.