The collected temporal bones were fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS; pH 7.4) for 2 h, and then harvested the CA under the microscope (SZX9, Olympus, Japan). The fixed specimens were immersed in PBS with 30% sucrose for 6 h, embedded in 5% agarose (type IX-A, Sigma-Aldrich, Ireland) and 20% sucrose in PBS, frozen in n-hexane (−60°C). These specimens were cut vertically into 15 μm thick sections from planum semilunatum to the center of the crista on a cryostat (Tissue-Tek Cryo3, Sakura Finetek, Japan; Kanda et al., 2008 (link)). At intervals of 45 μm, five sections including the center of CA were multiple-immunostained in each experiment and observed under a confocal microscope (A1+, Nikon, Japan; Supplementary Figure S2 ).
Agarose type 9 a
Manufactured by Merck Group
Sourced in United States
Agarose Type IX-A is a purified, low-electroendosmosis (EEO) agarose that is suitable for use in a variety of gel electrophoresis applications. It has a high gel strength and is commonly used for the separation and analysis of nucleic acids and proteins.
Automatically generated - may contain errors
5 protocols using agarose type 9 a
1
Cryosectioning and Immunostaining of the Cochlear Apparatus
2
Ultra-Low Gelling Agarose Resuspension
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An ultra-low gelling temperature (ULGT) agarose (Agarose Type IX-A, Sigma-Aldrich, St. Louis, MO, USA) that gels at temperature <17°C was used as a resuspending medium. A standard agarose (Agarose Type I-A, Sigma-Aldrich) that gels at <36°C was used as a re-embedding medium. Each agarose material was melted in boiling water at 3% (w/v). In order to preserve gelation quality of the agarose solutions, they were kept at 4°C with the cap fully tightened. When ready to be used, they were re-melted using a microwave oven. The re-melted ULGT agarose solution was then kept at room temperature while the re-melted standard agarose solution was kept in the oven set at 60°C to prevent premature solidification prior to use.
3
Agarose Pre-embedding of Cell Pellets
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Two types of agarose solutions were used in order to pre-embed the cell pellets. Three percent (w/v) ultra-low gelling temperature (ULGT) agarose (Agarose Type IX-A, Sigma-Aldrich, St. Louis, MO, USA) that has a gelling temperature of < 17°C was used as a resuspending medium. Three percent standard agarose (Agarose Type I-A, Sigma-Aldrich) with a gelling temperature of 34-38°C was used as a re-embedding medium. In order to preserve the gelation quality of the agarose solutions, they were kept at 4°C with the cap fully tightened. When ready to be used, they were re-melted using a microwave oven. The re-melted ULGT agarose solution was then kept at room temperature while the re-melted standard agarose solution was kept in an oven set at 60°C to prevent premature solidification prior to use.
4
Tissue Fixation and Cryoprotection Protocol
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Adult male and female piranha, head-and-tail-light tetra, medaka and zebrafish were deeply anesthetized with 0.02% MS-222 and fixed by perfusion with 4% paraformaldehyde (PFA) in PBS. For piranha and zebrafish, saline was perfused before the perfusion of the 4% PFA. After their brains were dissected out, they were post-fixed by 4% PFA in PBS for more than 4 hours. They were then immersed in 30% sucrose in PBS for more than 4 hours for cryoprotection. They were then embedded in 5% agarose (type IX-A; Sigma-Aldrich) and 20% sucrose in PBS, frozen in n-hexane (∼-60°C), and serial frontal sections were prepared at 25 μm thick on a cryostat.
5
Fabrication of Agarose-Collagen and Gelatin Hydrogels
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The AC hydrogels are made of agarose and collagen mixed into high glucose DMEM medium (Gibco). Stock agarose solutions were prepared by dissolving 3 wt/v% agarose Type IX-A with ultra-low gelling temperature (Sigma) in DMEM by autoclaving the mixture for 15 minutes at 100 °C the solution was then brought to 37 °C. Stock collagen solutions were prepared on ice immediately prior to use by neutralizing collagen type I solution (3.34 mg/ml rat tail collagen, Corning®) with dropping 1 M NaOH to bring the pH to 7.4. AC hydrogels were prepared by mixing agarose and collagen stock solutions with additional warm DMEM in appropriate volumes to create composite hydrogel.
Gelatin hydrogel was prepared by dissolving 10 wt/v% gelatin Type A with gel strength 300 (Sigma) in DMEM by autoclaving the mixture for 15 minutes at 100 °C. The solution was brought to 37 °C in a water bath.
Gelatin hydrogel was prepared by dissolving 10 wt/v% gelatin Type A with gel strength 300 (Sigma) in DMEM by autoclaving the mixture for 15 minutes at 100 °C. The solution was brought to 37 °C in a water bath.
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