Sp5 2 sted cw superresolution laser scanning confocal microscope

Wild-type C57/BL6 mice (between 1-month and 5-month-old) or adult rats were fixed by perfusion with 4 % paraformaldehyde containing 4 % sucrose. Immunofluorescence labeling was performed on 40 μm-thick sections. Floating brain sections were subjected to epitope retrieval by incubation in 10 mM trisodium citrate pH 6 and 0.05 % Tween20 for 30 min at 90°C prior to blocking. The sections were sequentially incubated with the indicated primary antibodies (Additional file 1: Table S1) and Alexa Fluor-conjugated secondary antibodies (Molecular Probes) diluted in Tris-buffered saline containing 1 % BSA and 0.25 % Triton-X 100, for 48 h and 2 h, respectively. Nuclei were labeled using Hoechst stain (Molecular Probes) before mounting the sections on slides. Confocal images were acquired on a Leica SP5 II STED-CW Superresolution Laser Scanning Confocal microscope and analyzed by ImageJ [44 ].

Brains were harvested from bigenic BACE1-YFP mice (3-month) and WT littermates were fixed overnight at 4°C in 4% paraformaldehyde and cryoprotected in PBS 30% sucrose/PBS. Immunofluorescence staining was performed on 40 μm coronal sections or 30 μm sagittal sections as described [30 (link),55 (link),56 (link)]. The following primary antibodies were used: BACE1 rabbit mAb D10E5 (1:125; Cell Signaling), synaptophysin pAb (1:75; R&D System), polyclonal MAP2 (1:250, SantaCruz), MAP2 mAb (1:500; gift of Dr. Lester Binder), and neurofilament mAb NFT160 (1:3,700; Sigma-Aldrich). Alexa Fluor 488-, 568-, or 647-conjugated secondary antibodies (Molecular Probes) were used for detection. Images were acquired on Zeiss LSM 510 META laser scanning confocal microscope (BACE1-YFP transgenic brain in Figures 1C and D) or Leica SP5 II STED-CW Superresolution laser scanning confocal microscope (Figure 1C), and processed using ImageJ software.