Vector elite abc kit rabbit

Brains were coronally sectioned to include both the cortex and hippocampus. Immunohistochemistry was performed on 40 μm free floating sections using a peroxidase based immunostaining protocol. In brief; endogenous peroxidase activity was quenched using 0.1% hydrogen peroxide, after which the membranes were permeabilized using 1% triton X100 in TBS. Non-specific binding was blocked using 1.5% normal goat serum, followed by incubation in primary antibody for p-mTOR (1:200 diltion, rabbit polyclonal pSer2481 antibody) and mTOR (1:200, rabbit polyclonal antibody) overnight at 4°C. Sections were washed in 1xTBS, and then incubated in secondary antibody (biotinylated goat anti-rabbit, 1:400, Vector Elite ABC kit rabbit, Vector Laboratories) for 90 min at 4°C. Finally sections were incubated in an avidin biotin enzyme reagent (Vector Elite ABC kit rabbit, Vector Laboratories). Immunostaining was visualised using a chromogen (Vector SG substrate, Vector Laboratories). Sections were mounted on slides and visualized using a Zeiss LSM-510 META multiphoton laser scanning microscope with a Zeiss 10× lens, representative 900 μm × 900 μm areas of cortex (4 regions of interest) and hippocampus (2 regions of interest) were imaged for analysis. The number of p-mTOR and mTOR positive cells per image was counted using the cell counter tool in Image J (NIH, USA).

Brains were dissected into left and right hemisphere, with one hemisphere used for histology. Brains were coronally sectioned to include both the cortex and hippocampus. Immunohistochemistry was performed on 40 μm free floating sections using a peroxidase based immunostaining protocol. In brief, endogenous peroxidase activity was quenched using 0.1% hydrogen peroxide, after which the membranes were permeabilized using 1% triton X100 in TBS. Non-specific binding was blocked using 1.5% normal goat serum, followed by incubation in primary antibody for Aβ (1:200, rabbit polyclonal, Invitrogen) overnight at 4°C. Sections were washed in 1xTBS, and then incubated in secondary antibody (biotinylated goat anti-rabbit, 1:400, Vector Elite ABC kit rabbit, Vector Laboratories) for 90 min at 4°C. Finally sections were incubated in an avidin biotin enzyme reagent (Vector Elite ABC kit rabbit, Vector Laboratories). Immunostaining was visualised using a chromogen (Vector SG substrate, Vector Laboratories). Sections were mounted on slides and visualized using a Zeiss LSM-510 META multiphoton laser scanning microscope with a Zeiss 10× lens, representative 900 μm × 900 μm areas of cortex (4 regions of interest) and hippocampus (2 regions of interest) were imaged for analysis. The number of Aβ positive puncta per image was counted using the cell counter tool in Image J (NIH, USA).