Western blotting was performed as described. Cells were washed once with ice-cold PBS and artificial cerebro-spinal fluid (Sigma-Aldrich, CA, United States). To normalize for protein load, membranes were reblotted (Re-Blot, Chemicon, United States) and incubated with an appropriate control antibody (see section “Results”). BSA method was applied to quantify the basic expression of loading proteins (Bio-Rad, United States). Blotting images were carried out using Image J (MIT, Boston, MA, United States). The representative image is taken from three repeated trials. Most western blot studies were in neurons, except IGF-1 analysis in astrocytes.
Artificial cerebrospinal fluid
Manufactured by Merck Group
Sourced in United States
Artificial cerebrospinal fluid is a sterile, isotonic solution that mimics the composition of the natural cerebrospinal fluid found in the human body. It is used in research and laboratory settings to maintain appropriate conditions for cells, tissues, or other biological samples.
Automatically generated - may contain errors
6 protocols using artificial cerebrospinal fluid
1
Protein Expression Analysis by Western Blot
2
Notch1 Activation in Intracerebral Hemorrhage
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As previous study reported [13 (link)], mice were anesthetized intraperitoneally with 4% phenobarbital sodium at a dose of 5 μl/g and implanted with an osmotic minipump (Alzet 1003D, Alza Corporation, CA, USA) in the ipsilateral lateral ventricle after ICH performed. Briefly, the Notch1 activator was prepared as previous described [13 (link)], Notch1-activating antibody (Sigma-Aldrich, St Louis, MO, USA), 10 μg/ml, was dissolved in the artificial cerebrospinal fluid (Sigma-Aldrich, St Louis, MO, USA) at a 1:4 dilution. Then the cannula was placed into the Tleft lateral ventricle (bregma: 0.6 mm posterior, 1.1 mm left lateral and 3.0 mm deep). Each mouse was infused for 3 days with 0.10 µl/h of Notch1-activating antibody by continuous intracerebroventricular infusion. Only artificial cerebrospinal fluid was used for the vehicle of Notch activator in Sham and ICH + Vehicle groups.
3
Mechanical and Optical Properties of Zebrafish Larvae
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Zebrafish larvae were sacrificed by an overdose of MS222 (0.2% in PBS; Sigma-Aldrich), embedded in low-gelling-point agarose (2.5% in artificial cerebrospinal fluid; Sigma-Aldrich) and cut into transverse sections using a vibratome. The artificial cerebrospinal fluid (aCSF) contained (in mM) 134 NaCl, 2.9 KCl, 1.2 MgCl2, 2.1 CaCl2, 10 HEPES buffer, and 10 glucose, adjusted to pH 7.8 with NaOH (81 (link)). A section thickness of 300 μm was used for indentation measurements as well as Brillouin microscopy. Refractive index (RI) measurements were performed using 200 μm sections. Zebrafish sections selected for indentation measurements were immobilized on tissue culture plastic with Histoacryl (B. Braun, Melsungen, Germany), which was sparsely applied between the tissue culture plastic and the agarose embedding at a distance from the zebrafish section. The sections were submerged in cooled aCSF during indentation measurements. For both Brillouin microscopy and RI measurements, the sections were immobilized by sandwiching them between two glass slides suitable for optical imaging and surrounded by aCSF. All vibratome sections were obtained from 4-day-old larvae.
4
Orexin-A and Dual Orexin Receptor Antagonist (DORA) Administration
5
Anesthetized Rat Brain Microinjection
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Each rat was anesthetized with an i.p. injection of achloralose (100 mg/kg) and urethane (500 mg/kg) dissolved in a borax solution (2%) (Sigma-Aldrich, Steinheim, Germany). Left femoral artery and vein were catheterized for the measurement of arterial pressure and for the administration of anesthetics, respectively. The catheter of the artery was connected to a pressure transducer and the signals were acquired using the MP-36 Biopac system (Biopac Systems, Santa Barbara, CA). Mean arterial pressure (MAP) was calculated as follows: MAP 5 diastolic pressure 1 pulse pressure/3. Body temperature was maintained at 368C using a feedbackcontrolled heating pad (Diagnostic & Research Instruments, Taipei, Taiwan).
After been fixed in a stereotaxic device, the rat's skull was exposed and a 2-mm-diameter hole was drilled on the right site for injection. The injection coordinates for the hypothalamic area were 3.0 mm caudal and 1.4 mm lateral to bregma and 8.5 mm deep from the surface of the cortex. The pretreatment chemical was unilaterally injected into the right hypothalamus 15 min before the injection of oxotremorine (1 nmol in 100 nL artificial cerebrospinal fluid, Sigma-Aldrich Chemicals). The injection site was verified with histological examination for the location of a dye injected at the end of the experiment. Each rat was used for only one experiment.
After been fixed in a stereotaxic device, the rat's skull was exposed and a 2-mm-diameter hole was drilled on the right site for injection. The injection coordinates for the hypothalamic area were 3.0 mm caudal and 1.4 mm lateral to bregma and 8.5 mm deep from the surface of the cortex. The pretreatment chemical was unilaterally injected into the right hypothalamus 15 min before the injection of oxotremorine (1 nmol in 100 nL artificial cerebrospinal fluid, Sigma-Aldrich Chemicals). The injection site was verified with histological examination for the location of a dye injected at the end of the experiment. Each rat was used for only one experiment.
6
Transplantation of OBNSCs in Spinal Cord Injury
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At 80% confluence OBNSCs were collected, and dissociated into single cells, and suspended in artificial cerebrospinal fluid (Sigma-Aldrich) at 30 000 cells per μl. A total of 5 μl of the cells were injected 0.5 cm rostral to injury site, and the needle was removed after remaining in place for another 5 min, and cyclosporine (Sandimmun, 10 mg kg -1 ) was injected beginning from one day before transplantation and finished on the day of killing.
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