Mice were transcardially perfused with 4% paraformaldehyde (PFA), 1 hour after in vivo ctDCS or sham treatment. Free-floating sections (40μm13 (link)) were sliced after overnight post-fixation of brains in 4% PFA at 4°C. pS6 Ser 240/244 (Cat# 5364, Cell Signaling Technology, Danvers, MA) was used as a primary antibody at 1:500 dilution overnight at 4°C. DyLight 488 conjugated goat antirabbit IgG (Cat# 111–485-144, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was used as secondary antibody at 1:500 dilution for 1 hour at room temperature. Sections were mounted with Vecta Shield mounting media with 4,6-diamidino-2-phenylindole and imaged using a Zeiss (Oberkochen, Germany) LSM 710 confocal microscope. pS6 density was analyzed by Volocity (Quorum Technologies, Lewes, UK) software and normalized by the pS6 density in the deep layer of sham ctDCS-treated slices. Superficial cortical layer pS6 density was measured from layer 2/3 to layer 5, whereas deep cortical pS6 density was measured from layer 5/6.
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Phosphorylated S6 Protein Imaging in Mouse Brain
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Corresponding Organization : Universitat Autònoma de Barcelona
Other organizations : Universidad Pablo de Olavide
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Variable analysis
independent variables
- CtDCS treatment
- Sham treatment
- PS6 density in superficial cortical layers (layers 2/3 to 5)
- PS6 density in deep cortical layers (layers 5/6)
- Mice were transcardially perfused with 4% paraformaldehyde (PFA) 1 hour after treatment
- Brains were post-fixed overnight in 4% PFA at 4°C
- Free-floating sections (40μm) were sliced
- PS6 Ser 240/244 primary antibody was used at 1:500 dilution overnight at 4°C
- DyLight 488 conjugated goat anti-rabbit IgG secondary antibody was used at 1:500 dilution for 1 hour at room temperature
- Sections were mounted with Vecta Shield mounting media with 4,6-diamidino-2-phenylindole
- PS6 density was analyzed using Volocity software and normalized by the pS6 density in the deep layer of sham ctDCS-treated slices
- Deep layer of sham ctDCS-treated slices
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