Sm2000r

Mice were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital (200 mg/kg; Somnopentyl; Kyoritsu Seiyaku). Mice were then perfused transcardially with 20 mL of phosphate-buffered saline (PBS; pH 7.4) at 20–25 °C, followed by perfusion for 3 min with the same volume of 4% paraformaldehyde (1.04005.1000, Merck Millipore) in 0.1 M phosphate buffer (PB; pH 7.4) at 20–25 °C. The brains were removed and post fixed overnight at 4 °C with the same fixative. After cryoprotection with 30% sucrose in 0.1 M PB, the brains were cut into 40-µm-thick parasagittal, coronal, or horizontal sections on a freezing microtome (SM2000R; Leica Biosystems). Sections were collected in six bottles containing 0.02% sodium azide in PBS and stored at 4 °C until use for free-floating immunostaining.

Fixed forebrains were cut coronally into 25-μm-thick sections using a freezing-sliding microtome (SM2000R; Leica Biosystems). Sections were collected in a 12-well plate and stored in cryoprotectant solution (35% ethylene glycol and 25% glycerol in PBS). Tissue sections were stained free-floating using polyclonal antibody CN6 (a follow-up antibody of CN3) directed against Aβ^{61 (link)}, and microglial staining was performed with a rabbit polyclonal anti-Iba1 antibody (1:500; Wako Chemicals). Sections were mounted on slides and co-stained with Congo red according to standard protocols.

The week following completion of behavioral testing, rats were euthanized with Fatal-Plus (Vortech Pharmaceuticals, Dearborn, MI, United States) and perfused (Masterflex Easy-Load Console Drive; 7518-00; Cole Palmer Instrument Co., Vernon Hills, IL, United States) with ice cold phosphate-buffered saline and 4% paraformaldehyde in 0.1M phosphate buffer (pH = 7.2). Brains were post-fixed at 4° overnight in 4% PFA and then transferred to a 30% sucrose solution (in 0.1M PBS) at 4° until slicing. Using a sliding microtome (Sm2000r Leica Biosystems, Wetzlar, Germany), whole brains were sliced coronally at 40 μm. Tissue slices will be kept in 96-well plates filled with an antifreeze solution (62.8 mg NaH₂PO₄, 2.18 g Na₂HPO₄, 160 mL dH₂O, 120 mL ethylene glycol, and 120 mL glycerol) at −20° until immunohistochemistry staining.

We determined the fidelity of anterograde transport of RGC axons to their target neurons in the SC by measuring the transfer of cholera toxin subunit B (CTB) conjugated to Alexa Fluor 488 (CTB; Molecular Probes, Eugene, OR, USA). We intravitreally injected 1.5 μl of 1 μg/μl solution of CTB using a Hamilton syringe (7643-01; Hamilton, Reno, NV, USA) in anaesthetized mice (2.5% isoflurane). Two days after CTB injection, we transcardially perfused mice as described earlier.^{15–19 (link),20 (link),1–5 (link)} Following perfusion, we dissected out brains and cryoprotected in 20% sucrose solution. We then obtained coronal midbrain sections (50 µm) using a freezing sliding microtome (SM2000R; Leica Biosystems, Buffalo Grove, IL, USA). We imaged CTB fluorescence in alternating sections of the SC using a Nikon Ti Eclipse microscope. We quantified CTB signal intensity using a custom ImagePro (ImagePro 7; Media Cybernetics, Rockville, MD, USA) macro as described previously.^{15 (link)}

Mice were deeply anesthetized with a mixture of ketamine (300 mg per kg) and xylazine (20 mg per kg) before they were transcardially perfused with 20 ml of ice-cold PBS followed by 20 ml of ice-cold 4% paraformaldehyde in PBS. Brains were isolated and postfixed in 4% PFA for 24 h, followed by incubation in 30% sucrose (in PBS, pH 7.5) for a further 48 h. Frozen brains were cut into 25 μm thick coronal sections on a sliding microtome (SM2000R, Leica Biosystems, Wetzlar, Germany) and collected in PBS. Immunohistochemistry was performed on free floating sections using the following antibodies diluted in PBS containing 5% normal goat serum and 0.5% Triton X-100: anti-Aβ 3552 (rabbit polyclonal antibody specific for Aβ 1–40 [40 (link)] (kindly provided by E. Kremmer, Ludwig Maximilians University, Munich, Germany); diluted 1:3000), anti-doublecortin (rabbit, DCX; 1:5000, abcam, ab18723), anti-Ki67 (rabbit, 1:500, abcam, ab15580). Appropriate secondary antibodies conjugated to Alexa 488 or 555 (1:1500) were used. Dense-core plaques were stained with Thiazinred (Sigma, T3272). Staining was done according to standard protocols. In brief, sections were washed 3 times in PBS and incubated in Thiazinred (2 μM solution in PBS) for 5 min at RT followed by 3 × 5 min washes with PBS.

Mice were transcardially perfused with 10 ml of ice-cold PBS followed by 10 ml of ice-cold 4% paraformaldehyde (PFA) in PBS (Roti-Histofix, Roth). Brains were isolated and post-fixed in 4% PFA for 24 h, followed by incubation in 30% sucrose (in PBS, pH 7.5) for a further 48 h. Frozen brains were cut into 25 μm thick coronal sections on a sliding microtome (SM2000R, Leica Biosystems, Wetzlar, Germany) and collected in 15% glycerol. Sections were blocked in 5% normal goat serum (NGS) in PBS with 0.5% Triton X-100. Afterward slices incubated overnight at 4°C with antibodies diluted in PBS containing 1% NGS against the following: anti-Aβ (mouse, 1:1,000, Covance, 6E10), anti-Iba1 (rabbit, 1:1,000, WAKO, 019-19741), anti-CD68 (rat, 1:500, BioRad, MCA1957), anti-GFAP (rabbit, 1:3,000, DAKO, Z033401-2), anti-C3 (rat, 1:200, abcam, ab11862), anti-PDGFRα (rabbit, 1:400, Cell Signaling, 3174), and anti-MBP (rabbit, 1:1000, abcam, ab40390). Corresponding secondary antibodies conjugated to Alexa 488 or 555 (1:1,000) were used. Sections were counterstained with DAPI (Sigma, D9542, 1:10,000) and mounted with a fluorescence mounting medium (DAKO, S3023).