Vt1200s vibratome

Hippocampal slices were meticulously prepared following the methodology outlined in our prior study [39 (link)]. In brief, mice were deeply anesthetized with pentobarbital (100 mg/kg i.p.) and transcardially perfused with a 4 °C slice-cutting solution comprising (in mM): 220 sucrose, 2.5 KCl, 1.3 CaCl₂-2H₂O, 2.5 MgSO₄, 1 NaH₂PO₄-2H₂O, 26 NaHCO₃, and 10 D-glucose, aimed at safeguarding neurons and preserving the functional connectivity of brain slices. Subsequently, mice were decapitated, and brains promptly extracted and kept in ice-cold cutting solution. Transverse hippocampal slices (300 μm thickness) were precisely sectioned by a VT1200S vibratome (Leica, Germany) in ice-cold cutting solution. The slices were then incubated in regular artificial cerebrospinal fluid (ACSF) containing (in mM): 126 NaCl, 3 KCl, 1.2 NaH₂PO₄-2H₂O, 1 MgSO₄, 2.0 CaCl₂-2H₂O, 26 NaHCO₃ and 10 D-glucose for 30 minutes at 33 °C and 1 hour at room temperature (25 ± 1 °C) before recording. All solutions underwent oxygenation with 95% O₂ and 5% CO₂.

Dorsal hippocampal slices (parasagittal, 250 µm) were prepared on a Leica VT1200S vibratome in ice-chilled slicing solution containing the following (in millimolar): 124 NaCl, 2.5 KCl, 26 NaHCO₃, 1 NaH₂PO₄, 10 glucose, 1 CaCl₂, 2 MgCl₂, and 1 kynurenic acid. Osmolarity was adjusted to ∼295 mOsM and pH set to 7.4 when bubbled with 5% CO₂.
For electrophysiology experiments, 300-µm parasagittal hippocampal slices from mice were prepared in ice-cold N-methyl-D-glucamine (NMDG)–based slicing solution (to reduce cell swelling), containing the following (in millimolar): 93 NMDG, 2.5 KCl, 20 HEPES, 30 NaHCO₃, 1.2 NaH₂PO₄, 25 glucose, 0.5CaCl₂, 10 MgCl₂, 5 sodium ascorbate, 2.4 sodium pyruvate, and 1 kynurenic acid. The osmolarity was adjusted to ∼300 mOsM and pH set to 7.4. Slices were immediately transferred to warmed (35 °C) slicing solution for 20 min and then to storage solution at room temperature containing the following (in millimolar): 92 NaCl, 2.5 KCl, 20 HEPES, 30 NaHCO₃, 1.2 NaH₂PO₄, 25 glucose, 2 CaCl₂, 1 MgCl₂, 5 sodium ascorbate, 2.4 sodium pyruvate, and 1kynurenic acid. The osmolarity was adjusted to ∼300 mOsm, and pH set to 7.4.

Timed pregnant Swiss Webster females (Taconic) were injected with EdU (50 μg per 1 g body weight) between E10.5-E13.5. At P30, pups were transcardially perfused with PBS and 4% paraformaldehyde. Brains were harvested, fixed overnight in 4% PFA at 4°C, and sectioned at 50 μm on a Leica VT 1200S Vibratome. For soma location quantification, sections were treated with a blocking solution of 5% normal donkey serum, 0.3% Triton-X, and PBS for 30 min at room temperature. Sections were then incubated in goat anti-ChAT (1:250 dilution, Millipore AB144P) overnight at 4°C. After PBS washes, sections were incubated in Alexa Flour 594 donkey anti-goat (1:1000 dilution) in the dark for 1 hr at room temperature, followed by additional washes in PBS. Sections were then treated with the Click-iT EdU kit for imaging Alexa Fluor 488 (ThermoFisher, C10337), followed by DAPI, additional PBS washes, and mounting of sections on slides.

Slices were prepared from P301S tau transgenic pups aged 7 days as we previously described (Miller et al., 2021 (link)). Brains were extracted and maintained in slicing medium (EBSS +25 mM HEPES) on ice. Brains were bisected along the midline using a sterile scalpel and the medial surface attached to the stage of a Leica VT1200S Vibratome using cyanoacrylate (Loctite). Sagittal slices of 300 μm thickness were removed and the hippocampus was dissected using sterile needles. Slices were maintained on membranes with 0.4 μm pore size (Millipore) in 6 well plates with 1 ml Slice Culture Medium as follows: 50% MEM with GlutaMAX (Gibco, 41090036), 18% EBSS (Gibco, 24010043), 6% EBSS + D-glucose, 1% penicillin-streptomycin (Gibco, 15140122), 0.06% nystatin (Gibco, 15340029), and 25% horse serum (Gibco, 26050070). Slices were maintained at 37°C and 5% CO₂ in a humid atmosphere.

In situ hybridization was carried out on whole-mount fixed planarians (WISH) and on sections as previously described (Nogi and Levin, 2005 (link); Umesono et al., 1999 (link)) either manually or by using the InsituProVSi hybridization robot (Invatis). Images were taken with a Leica M165 FC microscope. Fluorescence in situ hybridization (FISH) was performed as described (Cebrià and Newmark, 2005 (link); März et al., 2013 (link)) on whole animals and sections. For nuclear staining, Hoechst 33342 (Life Technologies) was used. For sections, the animals were passed through the in situ protocol until the first wash with Buffer 1 [100 mM maleic acid, 150 mM NaCl, 0.1% (v/v) Triton X-100]. Afterwards they were mounted in 3% agarose blocks and sectioned (70 μm) on a Leica VT 1200S vibratome before continuation of the protocol. FISH images were taken with a Zeiss laser-scanning microscope (LSM700) and processed and evaluated with Fiji (Schindelin et al., 2012 (link)). The LSM700 tile scan option, which automatically assembles tiled images to visualize larger structures, was applied for images in Fig. 2E; Fig. 3B,C; Fig. 4A; Fig. 5D; Fig. S4C,D; Fig. S6D; Fig. S10E; Fig. S11; Fig. S12 (sites of assembly are visible as thin lines). Primer sequences for in situ probe generation and references for marker genes are listed in Table S2.