Animals were euthanised and excised inner ears were fixed overnight in 2.5% glutaraldehyde in 0.1 M phosphate buffer (Sigma-Aldrich), then decalcified for 48 h in 4.3% EDTA in 0.1 M phosphate buffer (Sigma-Aldrich). Fine dissection was performed to reveal the organ of Corti, before osmium tetroxide (Agar Scientific)-thiocarbohydrazide (Fluka) (OTOTO) processing (adapted from Hunter-Duvar, 1978 (link)) was carried out. Samples were then dehydrated through increasing-strength ethanol solutions (Fisher Scientific) and critical point dried using an Emitech K850 (EM Technologies Ltd). Specimens were then mounted on stubs using silver paint (Agar Scientific) and sputter coated with platinum using a Quorum Q150T sputter coater (Quorum Technologies). Prepared cochleae were visualised with a JEOL LSM-6010 (Jeol Ltd) scanning electron microscope. Hair-cell counts were performed by counting the number of adjacent IHCs and OHCs to 20 pillar cells; for the analysis, the cochlea was divided into four separate regions (turns): apical (<90° from apex), mid-apical (90-180° from apex), mid (180-360° from apex) and mid-basal (360-540° from apex). Ears from at least three mice were analysed for each genotype at each turn and time point.
0.1 m phosphate buffer
Manufactured by Merck Group
Sourced in United States, Germany
0.1 M phosphate buffer is a common laboratory reagent used to maintain a specific pH range in various experimental and analytical applications. It is a balanced solution of sodium phosphate salts that provides a stable pH environment for biological samples, reactions, and other laboratory procedures. The precise ionic composition and concentration of this buffer help maintain a consistent pH, which is crucial for many experimental protocols. This product is widely used in fields such as biochemistry, cell biology, and analytical chemistry.
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Lab products found in correlation
Paraformaldehyde (pfa), by Merck Group (4 mentions)
Glutaraldehyde, by Merck Group (3 mentions)
Ketamine, by Merck Group (2 mentions)
Acetylcholinesterase assay kit, by Abcam (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Osmium tetroxide, by Merck Group (2 mentions)
Increasing strength ethanol solutions, by Thermo Fisher Scientific (1 mentions)
Silver paint, by Agar Scientific (1 mentions)
Quorum q150t sputter coater, by Quorum Technologies (1 mentions)
Lsm 6010, by JEOL (1 mentions)
Xylazine, by Merck Group (1 mentions)
Formalin, by Merck Group (1 mentions)
Ketamine xylazine, by Merck Group (1 mentions)
Sonoplus gm 70, by Bandelin (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Em grade glutaraldehyde, by Merck Group (1 mentions)
Ultra plus, by Zeiss (1 mentions)
Versamax plate reader, by Molecular Devices (1 mentions)
Leica em cpd030 critical point dryer, by Leica Microsystems (1 mentions)
Porcine skin gelatin, by Merck Group (1 mentions)
20 protocols using 0.1 m phosphate buffer
1
Scanning Electron Microscopy of Organ of Corti
2
TEM Analysis of Infected Macrophages
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For TEM analysis, infected macrophages were fixed with a solution containing 2% (wt/vol) paraformaldehyde (Merck, Ireland) and 2.5% (vol/vol) glutaraldehyde (Merck, Ireland) in 0.1 M phosphate buffer (Sigma–Aldrich, USA) at pH 7.4 for 1 h. After fixation, the infected macrophages were recovered using a cell scraper and were processed following conventional procedures (Lee et al., 2011 (link)). The sections were observed using a Jeol 1400 transmission electron microscope (Jeol, Japan).
3
Hippocampal Electrode Implantation for Kindling Epilepsy
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The rats were deeply anesthetized by a mixture of ketamine and xylazine (Sigma, England, 100/10 mg/ kg, intraperitoneal), fixed in a stereotaxic frame with their skulls exposed. A bipolar stimulating electrode and a monopolar recording electrode were twisted together and implanted in the hippocampal CA1 region of the right hemisphere at 2.4 mm posterior and 1.8 mm lateral to the bregma and 2.8 mm below the skull of each rat (19 ). In the kindling model of epilepsy the occurrence of after discharges (ADs) at the AD threshold is necessary for seizure progression (2 (link)). Therefore, it is essential to record ADs to ensure that the kindling procedure is developed correctly. The teflon-coated, stainless steel electrodes (A-M Systems, Inc., WA, USA, 127 μm in diameter) were insulated except at their tips. A monopolar electrode connected to a stainless steel screw was also positioned in the skull above the occipital cortex as a reference and/ or ground electrode. The electrode location was histologically confirmed in the animals at the end of the experiments. Rats were deeply anesthetized and perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (Sigma, England, pH=7.4). Then, their brains were removed and sectioned to verify electrode placement.
4
Corpus Callosum Myelination Analysis
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Animals were sacrificed by performing deep
anesthesia using ketamine/xylazine (100/10 mg/kg,
Sigma-Aldrich, Germany) and decapitated. The brains
were removed by craniotomy of the vertex, weighed
precisely and then immersed in the fixative solution
of formaldehyde and formalin (10%, Sigma-Aldrich,
USA) for more than 24 hours. After washing with
a 0.1 M phosphate buffer (Sigma-Aldrich, USA),
the brains were sectioned at the coronal plane and
using its natural cleavage plane, the corpus callosum
identified and dissected, removing samples of the
mid-body. After tissue processing, the blocks were
cut in sections of 5- and 6-ىm of thickness, and these
sections were used for Luxol Fast Blue (LFB) staining
and immunohistochemistry technique.
anesthesia using ketamine/xylazine (100/10 mg/kg,
Sigma-Aldrich, Germany) and decapitated. The brains
were removed by craniotomy of the vertex, weighed
precisely and then immersed in the fixative solution
of formaldehyde and formalin (10%, Sigma-Aldrich,
USA) for more than 24 hours. After washing with
a 0.1 M phosphate buffer (Sigma-Aldrich, USA),
the brains were sectioned at the coronal plane and
using its natural cleavage plane, the corpus callosum
identified and dissected, removing samples of the
mid-body. After tissue processing, the blocks were
cut in sections of 5- and 6-ىm of thickness, and these
sections were used for Luxol Fast Blue (LFB) staining
and immunohistochemistry technique.
5
Sulfur Metabolite Quantification in Cells
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The pellets containing 3.5–5 × 106 cells were suspended in 0.1 M phosphate buffer with a pH of 7.5 (Sigma-Aldrich, Darmstadt, Germany), maintaining the proportion of 1 million cells to 0.04 mL of the buffer, then sonicated for 3 × 5 s at 4 °C (Bandelin Sonoplus GM70, Berlin, Germany). Afterwards, they were centrifuged at 1600× g at 4 °C for 10 min and the supernatants were used to analyze the protein concentration, sulfane sulfur levels, and the activity of TST, MPST, and CTH.
In order to determine the low molecular weight sulfur-containing compounds by reversed phase high-performance liquid chromatography (RP-HPLC), the pellets were suspended in a 250 µL mixture consisting of 0.9% NaCl (Sigma-Aldrich, Darmstadt, Germany)/1 mM bathophenanthrolinedisulfonic acid disodium salt hydrate (BPDS, Sigma-Aldrich, Darmstadt, Germany)/70% perchloric acid (PCA, Polish Chemicals Reagents, Gliwice, Poland). The suspensions were then sonicated 3 × 5 s at 4 °C and centrifuged at 1600× g for 10 min (MPW-260R, MPW MED. INSTRUMENTS, Warsaw, Poland) to separate the sediment. The supernatants were collected and stored at −80 °C until RP-HPLC analyses.
In order to determine the low molecular weight sulfur-containing compounds by reversed phase high-performance liquid chromatography (RP-HPLC), the pellets were suspended in a 250 µL mixture consisting of 0.9% NaCl (Sigma-Aldrich, Darmstadt, Germany)/1 mM bathophenanthrolinedisulfonic acid disodium salt hydrate (BPDS, Sigma-Aldrich, Darmstadt, Germany)/70% perchloric acid (PCA, Polish Chemicals Reagents, Gliwice, Poland). The suspensions were then sonicated 3 × 5 s at 4 °C and centrifuged at 1600× g for 10 min (MPW-260R, MPW MED. INSTRUMENTS, Warsaw, Poland) to separate the sediment. The supernatants were collected and stored at −80 °C until RP-HPLC analyses.
6
Electron Microscopy Sample Preparation
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Living cells were fixed 24 h after treatments with 2% (w/v) paraformaldehyde (Sigma-Aldrich) and 3% (v/v) glutaraldehyde (EM grade, Merck, Darmstadt, Germany) in 0.1 M phosphate buffer (Sigma-Aldrich), following the protocol described elsewhere [14 (link)]. Briefly, the samples were post-fixed with OsO4 1%, washed several times with distilled H2O, and dehydrated in acetone, embedded in Eponate resin at 60°C for 48 h, and sectioned with an ultramicrotome. Finally, ultrathin sections placed in copper grids were contrasted with uranyl acetate and Reynolds lead citrate solutions, and examined using a Jeol 1400 (Jeol LTD, Japan) TEM equipped with a CCD GATAN ES1000w Erlangshen camera.
7
SEM Evaluation of EC and SC Co-cultures
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The scanning electron microscopy (SEM) was used to evaluate the attachment and morphology of the ECs and SCs in co-cultures at 1, 7 and 14 day time points. Additionally, the SCs were evaluated after 1-day pre-culturing. Briefly, the cells were washed with Dulbecco's phosphate-buffered saline and fixed with 5% glutaraldehyde (Sigma-Aldrich) in 0.1 M phosphate buffer (pH 7.4, Sigma-Aldrich) at room temperature for 48 h. Thereafter, the samples were dehydrated through a sequence of increasing concentrations (30, 50, 70, 80, 90, 95 and 100%) of ethanol for 5 min. For drying, the samples were transferred into a solution of 1 : 2 hexamethyldisilazane (HMDS, Sigma-Aldrich) and 100% ethanol (Altia Oyj, Helsinki, Finland) for 20 min following an incubation in 2 : 1 HMDS and ethanol for 20 min. Thereafter, the samples were dried twice in 100% HMDS for 20 min. Finally, the samples were allowed to evaporate in a fume overnight, gold sputtered and examined with SEM (Zeiss ULTRAplus, Oberkochen, Germany).
8
Acetylcholinesterase Activity Assay in Mice
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AChE activity in the brain tissue was determined using the Acetylcholinesterase Assay Kit (Abcam, Cambridge, UK). Brain tissue of mice from each group was homogenized in 0.1M phosphate buffer (Sigma-Aldrich Co.) and stored at −70℃ until analysis. The sample or standards and AChE reaction mixture were then incubated in a 96-well plate for 20 min at room temperature in the dark. Color alterations were read using a Versa max plate reader (Molecular Devices, Sunnyvale, CA, USA) at 405 nm.
9
Tissue Preparation for SEM Analysis
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The tissue specimens were fixed with 2% glutaraldehyde (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) in 0.1 M phosphate buffer (Sigma-Aldrich) at pH 7.4 at room temperature. The specimens were dehydrated through a gradual increase in the concentration of ethanol and amyl acetate (1st solution: 25% ethanol, 25% amyl acetate, 50% distilled water; 2nd solution: 35% ethanol, 35% amyl acetate, 30% distilled water; 3rd solution: 40% ethanol, 40% amyl acetate, 20% distilled water; 4th solution: 45% ethanol, 45% amyl acetate, 10% distilled water and; 5th solution: 50% ethanol and 50% amyl acetate; Merck Millipore, Darmstadt, Germany). Subsequently, the tissue specimens were dried at critical-point in a Leica EM CPD030 Critical Point Dryer (Leica Microsystems GmbH, Wetzlar, Germany) using liquid CO2. The fractured surface of the mandible was mounted on stub supports (Tousimis, Rockville, MD, USA) and platinum coated with a Plasma Sciences CrC-100 Turbo-Pumped sputtering system (Electron Microscopy Sciences, Hatfield, PA, USA), and observed using a Phenom G2 Pro scanning electron microscope (Phenom-World B.V., Eindhoven, The Netherlands).
10
Immunofluorescence Analysis of GAD67-GFP Mice DRGs
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GAD67-GFP mice (26 (link)) were transcardially perfused with 4% PFA under terminal anesthesia (sodium pentobarbital, 80 mg/kg). Lumbar DRGs were removed and stored in 0.1 M phosphate buffer (Sigma-Aldrich) followed by embedding in 10% porcine skin gelatin (Sigma-Aldrich), then postfixed in 4% PFA for 1 hour prior to sectioning. Thirty-five-micrometer DRG sections were cut using a vibrating microtome (Leica). Sections were washed once with 0.1 M PBS (Sigma-Aldrich) and blocked for 2 hours with blocking buffer (3% donkey serum in 0.1 M PBS; Sigma-Aldrich). Primary antibodies were diluted in 0.3% Triton X-100/PBS buffer before overnight incubation at 4°C. Detailed antibody information is given in Supplemental Table 5 . The following day, sections received a further 3 washes in PBS before incubation with secondary antibodies and/or GFP booster (Chromotek) for 4 hours at room temperature. Sections were washed with PBS 3 times and placed on microscope slides in Vectashield with DAPI (Vector Laboratories). Staining was visualized using a confocal fluorescent microscope (LSM700, Zeiss). Tissue preparation of electron microscopy is described in Supplemental Methods .
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