In each of the 4 groups, 3 mice were used for haematoxylin-eosin staining (H&E) at 3 days post-CCI. The animals were euthanized by intraperitoneally injecting sodium pentobarbital (65 mg/kg), then perfused transcardially with phosphate-buffered saline followed by 50 mL of 4% paraformaldehyde. The brains were quickly dissected from the mouse body and fixed in 4% paraformaldehyde at 4 °C for 48 h. Coronal sections, which were obtained using a vibratome (Leica VT 1000S, Wetzlar, Germany), should contain the entire hippocampus (–0 mm, –3.5 mm relative to bregma). Serial coronal sections (30 μm thick) for H&E staining (n = 3 per group) were cut by a cryostat (Leica CM 1950). At the same time, the colon tissue was excised and fixed in a 4% paraformaldehyde solution, dehydrated using ethanol and xylene, and embedded in paraffin. Moreover, 5 mm thick sections were cut for H&E staining. In addition, the injured cerebral cortex (respective n = 3) and colon tissue (respective n = 3) were quickly removed, weighed, frozen in liquid nitrogen, and then stored at −80 °C for Western blotting, and the cerebral cortex on the injured side (respective n = 3) was used for RNA-Seq. Furthermore, the faecal samples (respective n = 3) were quickly collected, weighed, frozen in liquid nitrogen, and then stored at −80 °C for SMRT sequencing.
Vt1000
Manufactured by Leica
Sourced in Germany, United States, France, United Kingdom, Israel, Japan, Switzerland, Canada, Belgium
The VT1000S is a vibratome, a precision instrument used for sectioning biological samples, such as tissues or organs, into thin slices for microscopic examination or further processing. The VT1000S provides consistent and accurate sectioning of samples, enabling researchers to obtain high-quality tissue sections for a variety of applications.
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Lab products found in correlation
Lsm 710, by Zeiss (36 mentions)
Lsm 780, by Zeiss (24 mentions)
Vectashield, by Vector Laboratories (22 mentions)
Fd rapid golgistain kit, by FD NeuroTechnologies (22 mentions)
Lsm 880, by Zeiss (16 mentions)
Lsm 710 confocal microscope, by Zeiss (16 mentions)
Paraformaldehyde (pfa), by Merck Group (15 mentions)
Vt1200, by Leica (14 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (14 mentions)
Fv1000, by Olympus (11 mentions)
Glutamax, by Thermo Fisher Scientific (11 mentions)
Triton x 100, by Merck Group (10 mentions)
Ab13970, by Abcam (10 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (10 mentions)
Lsm 700, by Zeiss (10 mentions)
Hoechst 33342, by Thermo Fisher Scientific (10 mentions)
Lsm 510, by Zeiss (10 mentions)
Aerrane, by Baxter (10 mentions)
Axio observer z1, by Zeiss (10 mentions)
Cm3050, by Leica (9 mentions)
1 652 protocols using vt1000
1
Comprehensive Analysis of Traumatic Brain Injury in Mice
2
Coronal Brain Slice Preparation
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Coronal brain sections of 350 μm in thickness were sectioned in a cold sucrose-based cutting solution containing (in mM): 195 sucrose, 10 NaCl, 25 NaHCO3, 25 glucose, 2.5 KCl, 1.25 NaH2PO4, 2 Na Pyruvate, 0.5 CaCl2, and 7 MgCl2 (pH 7.3) using a vibratome (Leica VT1000S) as described in previous studies57 (link)58 (link). Slices were permitted to recover in a submerged chamber at room temperature for at least 90 min before recordings, perfused with recording solution containing (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 2 CaCl2, 1 MgCl2, 25 glucose (pH 7.3) and continuously bubbled with 95% O2 and 5% CO2.
3
Golgi-Cox Staining of Hippocampal Dendrites
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To analyze the changes in the morphology of the dendritic spines in hippocampus, the Golgi-Cox staining was applied with minor modification as described by Hu et al. [12 (link)]. Briefly, brains stored at 37°C in dark place for two days in Golgi-Cox solution were sectioned at 200 μm in 6% sucrose with a vibratome (VT1000S, Leica, GER). All sections were collected on 2% gelatin-coated slides. Then slices were stained with ammonia for 60 min, washed with water for three times, followed by Kodak Film Fix for 30 min, and then washed with water, dehydrated, cleared, and mounted using a resinous medium. The pyramidal neurons in hippocampal region were imaged with a Nikon microscope (Nikon Eclipse 80i, Japan) using a 40x objective. The spines counted in the present study were on 2-3 stretches of the secondary dendrite about 10 μm in length. About 10–15 neurons from one animal were selected to quantify the spine density. Generally, brains were longitudinally cut into two halves and one hemisphere was processed for morphological staining and the other hemisphere was used to examine special proteins and genes expression.
4
Brain Slices from Wistar Rats
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Brain slice cultures of seven-day-old Wistar rats were prepared and maintained as previously described [41 (link)–42 (link)]. Animals were sacrificed and brains were removed and kept under ice-cold conditions. Frontal lobes and Cerebellum were dissected of the hemispheres. The remaining brain was cut into 350 μm thick horizontal slices using a vibratome (Leica VT 1000S, Bensheim, Germany). Brain slices were thereafter transferred onto culture plate insert membrane dishes (Greiner Bio One, Frickenhausen, Germany; pore size 0.4 μm) and subsequently transferred into six-well culture dishes (GreinerBioOne, Frickenhausen, Germany) containing 1.2 ml culture medium (MEM–HBSS, 2:1, 25% normal horse serum, 2% L-glutamine, 2.64 or 14.3 mg/ml glucose, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 10 μg/ml insulin–transferrin–sodium selenite supplement and 0.8 μg/ml vitamin C). The slices were cultured in humidified atmosphere (35°C, 5% CO2). The medium was changed on the first day after preparation and from that time forward every other day over a course of 7 days. Invasion fronts were determined by quantifying the tumor infiltrating area.
5
Hippocampal Slice Preparation from Transgenic Mice
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P21–P33 male and female C57BL/6J-Tg (Thy1-GCaMP6f) GP5.5 Dkim/J. mice (Jax 024276) mice were used to prepare paired hippocampal hemi-slices in accordance with a protocol approved by the Institutional Animal Care and Use Committee at Georgetown University Medical Center. Following deep isoflurane anesthesia, animals were rapidly decapitated. The whole brain was subsequently removed and chilled in iced (0°C) sucrose-based artificial cerebrospinal fluid (sACSF) containing (in mM) 252 sucrose; 3 KCl; 2 CaCl2; 2 MgSO4; 1.25 NaH2PO4; 26 NaHCO3; 10 dextrose; bubbled with 95% O2, 5% CO2. Hippocampal slices (480 μm thick) were cut in horizontal sections from dorsal to ventral brain with a vibratome (Leica, VT1000S). Slices were incubated in ACSF for at least 2 h before each experiment. ACSF used for maintenance and recording contained (in mM) 132 NaCl; 3 KCl; 2 CaCl2; 2 MgSO4; 1.25 NaH2PO4; 26 NaHCO3; 10 dextrose; bubbled with 95% O2, 5% CO2 at 26°C.
6
Transcardial Perfusion and Brain Sectioning
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Six (or nine) months after injection, mice were anesthetized with CO2 for 45 s, followed by transcardial perfusion with 20 ml of ice-cold PBS. Brains were extracted and postfixed for 48 h in 4% paraformaldehyde. Afterward, brains were stored in PBS containing 0.05% NaN3 and sectioned with a vibratome into 40-μm thick coronal sections (Leica VT1000S). To be able to identify ipsilateral and contralateral hemispheres relative to the tau injection after immunohistochemistry (IHC), the contralateral hemisphere received a small incision in the auditory cortex before sectioning.
7
Quantitative Analysis of Structural Plasticity in CCI
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Mice received a lethal dose of sodium pentobarbital (1.6 mg/g IP) 2 days postinjury. After loss of pain reflexes mice were transcardially perfused, first with heparinized (10 units/ml) saline for 1 min then 4% paraformaldehyde in Millonig’s buffer pH 7.4 for 20 min. Brains were dissected and then sectioned coronally at 60 μm using a vibratome (Leica VT1000S). Sections directly below the craniectomy (bregma level −0.6 to −2.4 mm) were collected in 24-well plates filled with Millonig’s buffer pH 7.4. To quantitatively assess for intrinsic AIS and extrinsic/synaptic perisomatic structural plasticity, for each animal, we labeled ankG, NaV1.6 and double-labeled glutamate decarboxylase-67 (GAD67) with PV in sections taken from a randomly selected and two adjacent wells, respectively, containing caudal S1BF (bregma level −1.5 to −2.0). This was done because of the consistency with which cFPI generates DAI within this well characterized area of neocortex, which was also the rational for choosing this region-of-interest (ROI) in our previously published electrophysiological studies (Greer et al., 2012 (link); Hånell et al., 2015a (link); Sun and Jacobs, 2016 (link)).
8
Culturing Mouse Cerebellar Slices
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Primary cerebellar cultures were prepared from C57BL/6J mice at postnatal day 10 as previously described [55 (link), 56 (link)]. Briefly, cerebella were embedded in 1% agarose and sagittally sectioned in ice-cold DPBS (Sigma‒Aldrich, G8769) supplemented with 5% glucose (Sigma‒Aldrich, G8769) at 300 μm using a Leica VT1000S vibrating microtome. Slices were immediately placed in 24-well plates on individual 0.4 μm 12 mm diameter Millicell-CM cell culture inserts (Millipore, PICM01250) and grown in 350 μL of cell culture medium containing 50% BME (Thermo Fisher, 21010046), 15% heat-inactivated horse serum (Thermo Fisher, 16050114), 25% Hank's solution (Sigma‒Aldrich, H4641), 0.5% glucose, 1% GlutaMAX™ Supplement (Thermo Fisher, 35050061), 1% penicillin‒streptomycin (Corning, 30-002-CI), N2, and 10 ng/mL PDGF-AA (Sigma‒Aldrich, P3076). The slices were incubated at 37 °C in 5% CO2. Half-volume media changes were made two days after plating. After 4 days, mouse cerebellar slices were treated with 10 µL of Nef or Ctrl EVs once daily for 2 or 4 days. The slices were fixed with 4% PFA for 15 min, delipidated in ice-cold 2.5% acid methanol for 15 min at − 20 °C, and subsequently processed for immunohistochemistry as described below.
9
Immunohistochemical Analysis of MCU
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Mice were anesthetized with 150 mg/kg sodium pentobarbital and transcardially perfused with 15–20ml of ice-cold 4% paraformaldehyde. Brains were dissected and post-fixed for at least 24 hours before sectioning 40 μm thick sections in the horizontal plane on a vibratome (Leica VT1000S). All brain sections immunostained with MCU were washed in 1X PBS-0.1% Triton X-100 (0.1% PBST) before they underwent antigen retrieval by boiling free floating sections for 5 min in 1 ml of nanopure water, followed by a permeabilization step in 0.1% PBST for 15 min at room temperature. All sections were then blocked for 1 hour in 5% Normal Goat Serum (NGS) in 0.1% PBST. Sections were incubated overnight at 4C (18–24 hours) with primary antibodies: rabbit-anti-MCU (1:2000, Novus Cat# NBP2-76948, Lot# H660681004, RRID:AB_2924913) and mouse-anti-RGS14 (1:500, NeuroMab Cat# 75–170, RRID:AB_2877352). Sections were then washed in 0.1% PBST several times and blocked for 30 minutes in 5% NGS in 0.1% PBST. Sections were incubated for 2 hours at room temperature in secondary antibodies (1:500, Invitrogen, AlexaFluors goat anti rabbit 546 Cat# A11035 and goat anti mouse 488 Cat# A11029) followed by several washes in 0.1% PBST and a final wash in 1X PBS.
10
In vivo Imaging of Seedling Roots and Exposed Sieve Tubes
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Seedling roots of the pSLI1:EYFP:SLI1 reporter line were imaged in vivo. Seedlings were grown on agar plates with 1% (w/v) agar and 0.5× Murashige Skoog (MS) medium at 22°C and 8‐hr:16‐hr L:D photoperiod. Seedlings were incubated at room temperature in 1 μM Mitotracker Deep Red (Thermo Fisher Scientific) 0.5× MS solution for 24 hr. For imaging of above‐ground plant tissue in 5‐to‐6‐week‐old pSLI1:EYFP:SLI1 reporter lines, sieve tubes were exposed using two different dissection methods. For longitudinal sections in the leaf midvein, mature leaves were cut at the base of the petiole and taped to a glass slide with the abaxial side up. With a sharp razor blade, a longitudinal section was removed from the petiole, starting with a cut at the base and ending with an ultrathin slice near the start of the leaf blade. The exposed area was directly submerged in distilled water to prevent desiccation. For transverse sections of the petiole and sections of the inflorescence stem and apex, tissues were cast in agar (5%) and sliced in 80 to 120 μm sections using a vibratome (Leica VT 1000 S).
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