Fluoview fv1000

Fluorescence images of brain slices were taken using a Zeiss fluorescent microscope (Axio Imager M2M). Confocal micrographs were acquired with either an Olympus Fluoview FV1000 or a Zeiss LSM880 AiryScan confocal microscope. For analysis, every 10th brain section approx. 1 mm anterior and posterior to the injection site were immunostained, with an interval of 250 μm per section. The definition of the CC was based on the mouse brain atlas (Franklin and Paxinos, 2019 ).
Total Aβ load was determined by calculating the relative areal fraction occupied with Aβ-positive staining (in %) in the CC using the image analysis software ImageJ (National Institutes of Health freeware, immunoblot analysis of injected brain homogenates). About four to seven animals per group and four to five sections per animal were analyzed.
Cell number was quantified by counting the number of positively labeled cells in the CC of the animals. This was mostly done in a semi-automated manner using custom-written macros. Since GFAP exclusively stains the processes of reactive astrocytes, the assessed cell number was substituted by the relative areal fraction occupied with the GFAP-positive signal (in %). To measure the thickness of the CC, six to seven animals per group and three to five sections per animal were analyzed. All analyses were conducted in a blinded manner.

After formaldehyde fixation (10% buffered formalin), brain tissues were paraffin embedded and sliced in 7 μm thick sections. DAB staining was performed as previously described [2 (link)]. Double immunofluorescence labelling was performed using Tyramide-FITC kit (NEL701A, Perkin Elmer), using a goat anti-rabbit antibody conjugated with biotin (Vector Laboratories, BA-1000) for SYNJ1 detection. Mouse monoclonal antibodies were detected using a goat anti-mouse antibody conjugated to Alexa 568 (A-11031, Invitrogen). Slides were mounted with Fluoromount-G (Southern Biotech) and immunofluorescence labelling was observed with an upright confocal microscope (Olympus Fluoview Fv1000) or with an Axiovert 200 M microscope (Zeiss) equipped with an ApoTome system (Zeiss). For quantitative analysis, SYNJ1 positive neurons and dystrophic neurites in hippocampal CA1–2 pyramidal layer were analysed at 40X images by thresholding analyses using NIH ImageJ as previously reported [58 (link)].

Formalin-fixed paraffin embedded sections (5 μm thickness) were baked at 60°C for 20 minutes, dewaxed in xylene for 1 hour, and rehydrated through an alcohol series. Antigen retrieval with 1X Reveal Decloaker (Biocare Medical) was performed using the capillary gapping method with a vegetable steamer for 35 minutes. Once sections were cooled and washed, they were blocked in Background Sniper (Biocare Medical) for 10 minutes. Primary antibodies, Chicken anti-Mouse/Human/Rat GFAP (Invitrogen PA1–10004) and Goat anti-Human/Mouse MPO (R&D AF3667) were diluted to 1 μl/mL and 0.5 μl/mL, respectively, in sniper and added to sections overnight at 4°C. Primaries were removed, and sections were washed in TBST twice before species-specific secondary antibodies Donkey anti-Chicken Ax594 at a 2 μl/mL concentration and Donkey anti-Goat IgG Ax488 at a 4 μl/mL concentration were added for 2 hours at room temperature. A TBS with 0.1% Triton X wash and distilled water wash performed, and then coverslips were mounted onto sections with Vectashield HardSet Antifade Mounting Medium with DAPI (Vector Laboratories) before imagining. Sections were imaged at 20x or 40x on an Olympus Fluoview FV1000 and neutrophils were counted on a Zeiss Axio Imager.M1 (20x and 40x objective).

Transfected cells adhered to round glass coverslips were washed 1X with PBS, fixed in 4% PFA-PBS for 20 min at RT, then washed 3 × 2 minutes in PBS. Fixed cells were permeabilized with PBT (PBS +0.1% Triton X-100) for 5 min. Non-specific reactions were blocked with PBT-N solution (PBT, 1% BSA, 5% FCS) for 1 h. Samples were incubated overnight (O/N) with rabbit polyclonal anti-GFP (1:500, Thermo Fisher Scientific A-6455) primary antibody at 4°C. Next day the samples were washed 3 × 2 minutes with PBT and incubated with the fluorescently labeled secondary goat anti-rabbit Alexa Fluor 488 antibody (1:600, Thermo Fisher Scientific A-11008) for 1 h at RT in dark. After washing 3X with PBT, Phalloidin Alexa Fluor 546 (1:40, Thermo Fisher Scientific, A22283) and DAPI (0.2 μg/mL, Sigma-Aldrich) in PBS were applied for 2 h in dark, at RT. Samples were washed 3X in PBS, and the coverslips were placed upside down in a drop of mounting medium (Fluoromount G, Thermo Fisher Scientific, 00-4958-02) on a microscope slide. Images were taken with Olympus Fluoview FV1000 (×40 oil immersion objective, 1.3 NA), Zeiss LSM 800 and Leica TCS SP5 (×63 oil immersion objectives, 1.4 NA) confocal microscopes.

Samples were fixed with 4% paraformaldehyde, washed with phosphate buffer with 0.2% Triton X-100, and staining as previously described [87 (link)]. Primary antibodies were used in the following dilutions: rabbit α-GFP 1:1000 (Invitrogen), chicken α-GFP 1:700 (Aves Labs), rat α-Ncad 1:20 (Developmental Studies Hybridoma Bank), mouse α-Bruchpilot 1:20 (Developmental Studies Hybridoma Bank), mouse α-CD2 1:1000 (Serotec), mouse α-Dac 2–3 1:20 (Developmental Studies Hybridoma Bank), rabbit α-β galactosidase 1:800 (Invitrogen), mouse α-β galactosidase 1:800 (Promega), rat α-Bab2 1:1500 (Frank Laski), rabbit α-Bar-H1 1:100 (Tetsuya Kojima). The following secondary antibodies were used: Alexa 488 goat α-rabbit 1:1000, Alexa 488 goat α-chicken 1:1000, goat α-mouse-Cy3 1:100, goat α-rat-Cy3 1:200, goat α-rabbit-Cy3 1:200, Alexa 568 goat α-mouse IgG highly cross-adsorbed 1:300, Alexa 647 goat α-rat 1:200, Alexa 633 goat α-mouse 1:200, Alexa 647 goat α-mouse 1:200. Confocal images were taken by an Olympus Fluoview FV1000 or Zeiss LSM 510 (Light Microscopy Core Facility).