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The brain sections were exposed on phosphor imaging plates (SR 2025, FujiFilm, Japan) overnight.
In vitro- or ex vivo-labeled brain sections were exposed to phosphor films (Perkin Elmer Multisensitive, Medium MS) and were read using the Cyclone Phosphor Imaging System (Packard Instruments, Meriden, CT, USA).
After being washed, the brain sections were exposed to Kodak Hyperfilm™ MP film.
Brain sections were exposed to a Kodak® BioMax MR film (Sigma-Aldrich, Poole, UK) for 3 8 days, and optical density for the area of interest was measured using an image analysis software MCID-M4, ver 3.0, rev.1.5 (Interfocus Imaging Ltd ,Cambridge, UK).
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Since fixation and embedding may alter tissue volume [ 54, 55], care was taken to ensure all brains and sections were exposed to the reagents for the same length of time.
Slides containing PAG sections were exposed for 4 weeks.
The sections were exposed to RUFY3 (1 :��100) and anti-PAK1 (1 : 200) 4 °C overnight.
Hybridized sections were exposed to autoradiographic film for 7 days.
Sections were exposed to phosphorimaging plates (GE Healthcare, Piscataway, NJ, USA).
" As each part of the body was washed, that small section was exposed, then covered.
Brain sections were incubated in 70% formic acid for 10 min to expose the epitope prior to incubation with blocking solution (PBS containing 0.5% Triton X-100, 0.1% BSA and 2% normal goat serum) for 2 h at room temperature.
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