Germline-GCaMP6F pups (P0-P6) were anesthetized with isoflurane; the forebrain was exposed and removed, enabling isolation of the hindbrain and spinal cord in chilled artificial cerebrospinal fluid (aCSF) made up of (in mM) 128.0 NaCl, 3.0 KCl, 1.2 CaCl2, 1.0 MgSO4, 21.0 NaHCO3, 0.5 NaH2PO4, and 30.0 glucose, equilibrated with 95% O2–5% CO2. The hindbrain was transected at the level of the sensory portion of the trigeminal nerve, just rostral to the facial nucleus (VIIn). The preparation was then mounted with the dorsal side down on an attachment permitting sectioning at compound angles [57 (link)], using the basilar artery to reproducibly align the preparation along the midline of the chuck. A sagittal section was cut at the lateral edge of the VIIn, visible at the surface of the preparation, at 17.7° ventrodorsal tilt and 3.7° rostrocaudal tilt relative to the midline, to expose the VRC along its major axis. The preparation was mounted on the face of a Sylgard (Dow Corning, Midland MI) block cut at the ventrodorsal tilt angle, so that the sagittal face of the preparation, aligned with the top edge of the Sylgard block, was perpendicular to the light path. A razor-blade fragment embedded in the Sylgard block enabled the preparation to be stabilized in the recording chamber by the placement of a 10-mm neodymium magnet on the undersurface of the recording chamber.
Sylgard
Manufactured by Dow
Sourced in United States, United Kingdom, Germany
Sylgard is a silicone-based encapsulant and potting compound developed by Dow. It is designed to protect and insulate electronic components and devices. Sylgard provides superior electrical insulation, thermal conductivity, and resistance to environmental factors such as moisture, chemicals, and vibration.
Automatically generated - may contain errors
Lab products found in correlation
Fv1000mp, by Olympus (4 mentions)
Sytox green, by Thermo Fisher Scientific (3 mentions)
Tris base, by Merck Group (2 mentions)
Digidata 1440a, by Molecular Devices (2 mentions)
Microforge, by Narishige (2 mentions)
Matrigel, by Thermo Fisher Scientific (2 mentions)
Sylgard 184, by Dow (2 mentions)
5500 atomic force microscope, by Keysight (2 mentions)
Hsq resist, by Dow (2 mentions)
Dimension 3000, by Veeco (2 mentions)
Hbss medium, by Thermo Fisher Scientific (2 mentions)
Nucleopore track etch membrane, by Cytiva (2 mentions)
Glucose 1m, by Merck Group (2 mentions)
Superfrost, by Thermo Fisher Scientific (2 mentions)
Silicone vacuum grease, by Dow (2 mentions)
Dumont 5 forceps, by Fine Science Tools (2 mentions)
Axopatch 200b patch clamp amplifier, by Molecular Devices (2 mentions)
Triton x 100, by Merck Group (2 mentions)
880 airyscan confocal microscope, by Zeiss (2 mentions)
Permafluor, by Thermo Fisher Scientific (2 mentions)
113 protocols using sylgard
1
Isolated Hindbrain and Spinal Cord Preparation
2
Tadpole Touch Response Quantification
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The response of the tadpoles to a touch stimulus (hair, 10 μm tip) applied to the head was analysed using high‐speed video recording (Exilim EX‐F1; Casio, Tokyo, Japan) with 300 frames s–1. For this, the animals were placed in a small Petri dish (diameter 8 cm) base‐filled on one side with a layer of Sylgard (Dow Corning, Midland, MI, USA) in an upright (dorsoventral) position within a groove cut into the edge of the Sylgard so that the head and cement gland were unrestrained. An array of LED lights provided even illumination from below. The videos were cut and analysed using ImageReady video editing software (Adobe Systems Inc., San Jose, CA, USA) and ImageJ image processing software (NIH, Bethesda, MD, USA).
3
Dissection and Pharmacological Manipulation of Retzius Neurons
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All experiments were done on electrically coupled Retzius (R) neurons from midbody ganglia (ganglia 7 to 16). Before dissection, leeches were anesthetized on ice cooled water for at least 10 min. Segmental ganglia were dissected and removed from the animal as previously described69 (link) and pinned, ventral side up to a superfusion chamber coated with Sylgard (Dow Corning). Dissection was carried out in leech Ringer solution composed of (in mM): NaCl, 115; KCl, 4; CaCl2, 1.8; MgCl2, 1.5; glucose, 10; tris-(hydroxymethyl)-aminomethane (Tris) maleate, 4.6; Tris base, 5.4 (all Sigma), buffered to pH 7.4. For pharmacological experiments, the ventral glial sheath covering the ganglia was opened with a fine microscissor to ensure the direct exposure of the R cells to the drugs in the external solution.
4
Spatial Calcium Imaging of Intestinal Interstitial Cells
5
Bovine Ampullae Inflammation Analysis
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Qualitative and quantitative analyses in bovine ampullae showing mild, moderate or severe inflammation (n = 45) as well as in healthy controls (n = 10) were performed by digital live cell imaging. 1 cm part of the ampulla of each sample was cut and opened longitudinally. Specimens were pinned onto a Delta T dishes (Bioptechs Inc., PA, USA) coated with Sylgard® (Dow Corning, MI, USA). All samples were maintained at 37.5 °C using a stage heater (Delta T Controller, Bioptech Inc., USA) and an objective lens heater (Delta T Controller, Bioptech Inc., USA) attached to the microscope. Reference temperature was monitored prior to all recordings. For sperm-oviduct interaction and average survival time, 600,000 sperm (30 μl) were coincubated with the ampulla for 10 mins at 37 °C and submerged with sperm Talp before analysis. Imaging was performed with a fixed stage microscope, upright Olympus microscope (BX51WI) with water immersion dipping objectives equipped with the bright-field long-distance immersion objectives UMPLFLN 10xW, UMPLFLN 20xW and UMPLFLN 40xW (Olympus, Hamburg, Germany). Images and videos were documented with a SUMIX Mx7 camera (Summix, CA, USA).
6
Microfluidic Device Fabrication via Replica Molding
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The microfluidic layouts (Fig. 1a ) were designed by means of a CAD software (AutoCAD, Autodesk Inc.) and the corresponding master molds were realized through photolithography techniques43 . A thin microfluidic layer (thickness 1 mm) was obtained by replica molding of polydimethylsiloxane (PDMS; Sylgard® Dow Corning, Michigan, USA). Briefly, liquid PDMS was cast on the mold in ratio 10:1 w/w (pre-polymer to curing agent), degassed and cured at 80 °C for 3 h. The obtained PDMS microstructured layer was permanently bonded facing up to a flat PDMS slab (thickness 5 mm), upon 1 min of air plasma treatment (Harrick Plasma, NY, USA), thus closing the channels. Input and output ports were obtained through a 0.5 mm biopsy puncher (Harris Uni-CoreTM). The chip was finally permanently bonded to a glass coverslide (diameter 50 mm, thickness 150 μm; VWR International Inc., PA, USA) upon an additional 1 min exposure to air plasma (Fig. 1b ). External connections were realized through tygon tubings (ID = 0,02”; Qosina, NY, USA) and stainless steel couplers (23 gauge; Instech Laboratories Inc., PA, USA).
7
Gluteus Maximus Muscle Dissection Technique
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Once anesthetized, mice were placed under a stereomicroscope. The left gluteus maximus area of the mouse was shaved and cleaned to remove the fur. The gluteus maximus muscle was chosen over the cremaster muscle, as used in other experiments such as Payne et al. (2003) [18 (link)], because the cremaster is only found in males and we performed these experiments in both males and females. The skin covering the muscle area was cut away and the proximal edge of the gluteus maximus muscle cut parallel to the spine and reflected away from the mouse ensuring preservation of the vascular supply to the tissue. The muscle was then positioned to lay flat and the proximal edge pinned down on a Sylgard (Dow Corning, Midland, MI, USA) board. Physiological saline solution (PSS) made of bicarbonate (18.0 mM) and basic salt solution (131.9mM NaCl, 4.7 mM KCl, 2.0 mM CaCl2•2H2O, 1.17 mM MgSO4•7H2O) at pH 7.4 was superfused over the muscle tissue to mimic physiological conditions. The solution was kept at a temperature of 37 °C using a heated perfusion reservoir (Radnoti, USA).
8
Cephalopod Pedal Nerve Stimulation
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Animals were anesthetized by injecting 333 mM MgCl2 (~50% of body weight). Pedal ganglia were removed maintaining the innervation of the foot muscles. This innervation extends from the posterior edge of the mantle cavity to the anterior tentacles and parts of the body wall. Preparations were pinned to a 2-chamber dish lined with Sylgard (Dow Corning) (Fig. 3 A). To facilitate the pinning, we only preserved the innervation of the experimental (mostly right) side of the preparation. The pedal nerves on the other side were severed. The foot muscle was situated in the larger of the 2 chambers, whereas the pedal ganglion was pinned to the smaller chamber, which had a higher Sylgard floor.
We cannulated the pedal artery, and foot muscles were continuously perfused with fresh ASW at ~0.5 ml/min throughout the experiments. Perfusion began immediately after the preparation was isolated. Experiments were initiated after 2 to 3 h (i.e., washout of the anesthetic). To monitor muscle contractions elicited by either P9 stimulation or motoneuronal stimulation, the edge of foot was pinned with multiple pins, and the middle of the foot was attached to a force transducer (Isotonic Transducer “60–3000,” Harvard Apparatus) (see Fig.3 A). Preparations were maintained at 14 to 16 °C.
We cannulated the pedal artery, and foot muscles were continuously perfused with fresh ASW at ~0.5 ml/min throughout the experiments. Perfusion began immediately after the preparation was isolated. Experiments were initiated after 2 to 3 h (i.e., washout of the anesthetic). To monitor muscle contractions elicited by either P9 stimulation or motoneuronal stimulation, the edge of foot was pinned with multiple pins, and the middle of the foot was attached to a force transducer (Isotonic Transducer “60–3000,” Harvard Apparatus) (see Fig.
9
Synthesis and Characterization of Silver Nanoparticles
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A silver (I) nitrate particle sample was purchased from Daejung Chemical & Metals (Shiheung-City, Korea). Sodium sulfate powder was purchased from Junsei Chemical (Tokyo, Japan). Phosphate buffered saline (PBS) (pH 7.4, 10 mM) solution used as the electrolyte in this study and triton X-100 solution were obtained from Sigma-Aldrich (St. Louis, MO, USA). Single layer graphene oxide (500 mg/L) dispersed in water was purchased from Graphene Supermarket (Calverton, NY, USA). Sylgard (Dow Corning, Midland, MI, USA) 184 silicone elastomer curing agent and Sylgard 184 silicone elastomer base were acquired from Dow Corning (Midland, MI, USA) for polydimethylsiloxane (PDMS) preparation. All aqueous solutions were prepared using deionized water (DIW) from a Millipore Milli-Q water purifier operating at a resistance of 18 MΩ/cm.
10
Acute Rat Cochlear Recordings
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Recordings were performed on acutely excised cochlear preparations from Sprague Dawley rats (Charles River Laboratories) at room temperature (22–25°C). Recording pipettes were fabricated from 1 mm borosilicate glass (WPI). Pipettes were pulled with a multistep horizontal puller (Sutter), coated with Sylgard (Dow Corning) and fire polished. Drug application was mediated by whole-bath perfusion or a gravity-driven flow pipette (100-µm-diameter opening) placed near the row of IHCs and connected with a VC-6 channel valve controller (Warner Instrument).
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