Vt 1000s microtome

All animal experiments were performed according to protocols approved by the Animal Care and Use Committee the University of North Carolina at Chapel Hill. Striatal slices were prepared from 14 to 24-day-old male and female C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME). Mice were euthanized using isoflurane, decapitated, and brains were placed into ice-cold sucrose buffer containing (in mM): 254 sucrose, 10 D-glucose, 26 NaHCO₃, 2 CaCl₂, 2 MgSO₄, 3 KCl, and 1.25 NaH₂PO₄, saturated with 95% O₂/5% CO₂, at pH 7.4, 300 mOsm. Brains were cut into 300 μM coronal sections through the striatum using a VT 1000S microtome (Leica, Deerfield, IL) and placed into a holding chamber at 33°C for 30 min in a mixture of 50% sucrose saline and 50% artificial cerebrospinal fluid (aCSF) containing (in mM): 128 NaCl, 10 D-glucose, 26 NaHCO₃, 2 CaCl₂, 2 MgSO₄, 3 KCl, and 1.25 NaH₂PO₄. Slices were then maintained at room temperature in aCSF bubbled continuously with 95% O₂/5% CO₂.

OMP-spH brains were fixed in 4% PFA in 1× PBS overnight and serial coronal sections (50 μm) of the OBs were taken using a Leica VT1000S microtome. Free-floating sections were mounted on VWR Superfrost Plus Micro Slides with VECTASHIELD Mounting Medium, which were then coverslipped and sealed with nail polish.
P2-lacZ and M72-RFP brains were processed for cryo-sectioning. Fresh (unfixed) brains were dissected out of the skull and transferred to a 30% sucrose solution in distilled water at 4 °C until they sank to the bottom of the container. Brains were then embedded in Tissue-Tek optimum cutting temperature (O.C.T.) in a cryo-mold and rapidly frozen on dry ice. Samples were stored at –80 °C and placed in cryostat chamber at –16 °C for 1 h prior to sectioning to achieve temperature equilibration. Serial coronal sections (16 μm) of the OBs were collected onto VWR Superfrost Plus Micro Slides. VECTASHIELD Mounting Medium with DAPI was placed on M72-RFP slides, which were then covered with a glass coverslip and sealed with nail polish. Immunohistochemistry, as described below, was performed on P2-lacZ sections before slides were covered with a glass coverslip with VECTASHIELD Mounting Medium with DAPI and sealed with nail polish.

Horizontal hippocampal-entorhinal cortex slices were prepared from Cntnap2 KO mice and age-matched WT sibling controls. Following deep anesthesia with isoflurane, mice were euthanized by decapitation. The brain was rapidly dissected and immersed in ice-cold oxygenated (95% O₂/ 5% CO₂) high-sucrose artificial cerebrospinal solution (sACSF) containing (in mM) 220 sucrose, 3 KCl, 26 NaHCO₃, 10 dextrose, 2 MgSO₄, 1.25 NaH₂PO₄ and 1 CaCl₂. 320 mm thickness slices were made using a VT 1000S microtome (Leica) and immediately after transferred to an incubation chamber with artificial cerebrospinal fluid (ACSF) containing (in mM) 124 NaCl, 26 NaHCO₃, 10 dextrose, 3 KCl, 1 CaCl₂, 2 MgSO₄, and 1.25 NaH₂PO₄, and maintained at 35°C for 30 min, and thereafter at room temperature for at least 40 min before recording. Ex vivo recordings were performed in slices from P90–110 WT and Cntnap2 KO mice, or P60–90 WT PV-tdt (+/‒) and Cntnap2 KO PVtdt (+/‒) mice.

After harvesting the root nodules from the legumes, nodules were cut longitudinally with a razor blade and embedded in 4% agarose. After polymerization under vacuum conditions for 15min at room temperature, thin sections of 80–90μm were prepared with a Leica VT1000S Microtome. Sections were incubated in 100-fold diluted SYBR® green I nucleic acid stain (Sigma-Aldrich, Darmstadt, Germany) for at least 30min at 4°C. Morphology of the root nodules was investigated with an Olympus SZX12 stereo microscope equipped with Olympus DF PLAPO 1XPF objective lens (Olympus, Shinjuku, Tokio, Japan) coupled with a Zeiss Axiocam 503 color camera (Zeiss, Oberkochen, Germany).

Embryonic and postnatal livers were dissected and fixed for 2 h or 24 h, respectively, in 10% neutral-buffered formalin (Sigma-Aldrich) at 4°C. Postnatal livers were embedded in 4% low melting point agarose (Bio-Rad Laboratories) and sectioned at 100 μm using a Leica VT1000S microtome. E10.5 and E11.5 samples were equilibrated with 30% sucrose, embedded in OCT compound (VWR, 361603E) and frozen on dry ice in preparation for sectioning with a Leica CM-3050S cryostat at 50 µm. To reduce nonspecific staining and permeabilize the sample, samples were incubated with a 2% donkey serum, 1% Triton and 5% DMSO in PBS solution overnight at 4°C. Primary antibodies were then applied at appropriate dilutions for 48 h at 4°C; for details refer to Table S3, part 1. Samples were washed and secondary antibodies applied at a dilution of 1:250 for 48 h at 4°C. Nuclei were counterstained with Hoechst 33342 (1:1000, Invitrogen) for 30 min at room temperature. Details of secondary antibodies are provided in Table S3, part 1.

Free-floating sections were essentially prepared as described before [21 (link)]. Mice were transcardially perfused with 4% paraformaldehyde (PFA). Subsequently, the spinal cord was removed and post-fixed in 4% PFA for 2 h at RT. After embedding in 5% agarose, 40 μm thick, free-floating sections were cut on a Leica VT1000 S microtome and collected in 0.1 M phosphate buffer pH 7.4. After incubation for 2 h at RT with blocking solution [10% donkey serum, 0.3% Triton X-100 and 0.1% Tween-20 in Tris-buffered saline (TBS)], the sections were incubated for 72 h at 4 °C with primary antibodies in 1:10 diluted blocking solution (1% donkey serum, 0.03% Triton X-100 and 0.01% Tween-20 in TBS). After washing three times for 10 min in TBS-Tween (TBS-T) at RT, sections were incubated for 2 h at RT with the appropriately fluorophore-conjugated secondary antibodies, washed again in TBS-T and finally mounted with Fluor Save (Merck-Millipore). The following primary antibodies were used: polyclonal goat anti-ChAT (MAB144P, Millipore; 1:1000) and polyclonal rabbit anti-TDP43 (10782–2-AP, Proteintech; 1:1000).