Mice were deeply anesthetized using intraperitoneal injection of urethane and perfused transcardially with 20 mL of Hanks’ Balanced Salt Solution (HBSS; 14025076, Life Technologies, USA) supplemented with heparin (10 units/mL), followed by perfusion with 4% formaldehyde in 0.1 M phosphate buffer (PB; pH 7.4) and postfixation in the same fixative for 16–20 h at 4°C. For subsequent in situ hybridization experiments, the brain blocks were cut into 40-μm-thick coronal sections using a freezing microtome (ROM-380, Yamato, Japan) after cryoprotection with 30% sucrose in PB. For the other experiments, the brain blocks were cut into 80-μm-thick coronal sections on a vibratome (VTS1200S, LEICA, Germany).
Rom 380
Manufactured by Yamato Scientific
Sourced in Japan
The ROM-380 is a scientific instrument designed for optical rotatory dispersion measurements. It is capable of determining the angle of rotation of polarized light as it passes through a sample. The device provides accurate and reliable data on the optical activity of various substances.
Automatically generated - may contain errors
Lab products found in correlation
Somnopentyl, by Kyoritsu Seiyaku (2 mentions)
Mab318, by Merck Group (2 mentions)
Vts1200s, by Leica (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Tcs sp5 confocal scanning microscope, by Leica (1 mentions)
Avertin, by Fujifilm (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Neutral buffered formalin, by Nacalai Tesque (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
6 protocols using rom 380
1
Perfusion and Brain Sectioning for In Situ Hybridization
2
Quantifying Thalamocortical Axon Innervation
3
Histological Analysis of Eye Tissue
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At 4, 8, and 12 weeks after surgery, animals were anesthetized by intraperitoneal administration of ketamine and xylazine, and sacrificed immediately by cutting the descending aorta and inferior vena cava. Whole eyes containing upper and lower eyelids were resected, and fixed by immersion with 10% neutral buffered formalin (Nacalai Tesque Inc., Kyoto, Japan) for about 1 week. Then, the tissues were embedded in paraffin using standard procedures. Approximately 3-µm thickness serial sections were cut using a sliding microtome (ROM-380; Yamato Kohki Industrial Co., Ltd., Saitama, Japan), placed on glass slides (Fine Frost Micro Slide Grass; Matsunami Glass Ind., Ltd., Osaka, Japan). The sections were deparaffinized and rehydrated, and then stained with hematoxylin-eosin (H&E) using standard procedures.
4
Visualizing Dopaminergic Neurons in VTA and SNc
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After completion of electrophysiological recordings, small electrical lesions were made through the electrodes (20 µA for 20 s, Stimulus Isolator A365, WPI). Birds were deeply anesthetized with an overdose of pentobarbital sodium (Somnopentyl, Kyoritsu Seiyaku) and perfused with 4% paraformaldehyde (PFA). Brains were dissected out, and post-fixed overnight in 4% PFA followed by 30% sucrose in phosphate-buffered saline. Sagittal brains sections (40 µm in thickness) were made with a freezing microtome (ROM-380, Yamato Kohki Industrial), and stained with an antibody against tyrosine hydroxylase (TH, MAB318, Merck Millipore) to visualize dopaminergic neurons in VTA and SNc. The electrode track was verified under a microscope.
5
Tracing Midbrain Dopaminergic Neurons
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After electrophysiological recordings, electrical lesions were made (20 μA, 20 s, Stimulus Isolator A365, WPI) or Fluoro-Ruby was deposited using an iontophoresis pump (BAB-501, Kation Scientific). The birds were deeply anesthetized with an overdose of pentobarbital sodium (Somnopentyl, Kyoritsu Seiyaku) and perfused with 4% paraformaldehyde (PFA), and brains were dissected out. The brains were post-fixed overnight in 4% PFA followed by 30% sucrose in phosphate-buffered saline. Sagittal brains sections (40 μm in thickness) were made with a freezing microtome (ROM-380, Yamato Kohki Industrial). Midbrain dopaminergic neurons were stained with an antibody against tyrosine hydroxylase (TH, MAB318, Merck Millipore), and the recording electrode track or injection site of Fluoro-Ruby was verified (Figure 1D ).
6
Histological Analysis of Bone Samples
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At time intervals, the bone specimens were fixed in 10% neutral phosphate-buffered formalin solution for 1 week and decalcified with 10% formic acid for 4–6 weeks and rinsed overnight with running water. All the samples were dehydrated using ethanol (50%–100%) and processed for paraffin embedding (Vacuum Rotary, VRX-23, Mitsubishi, Tokyo, Japan). Later, a 5 µm thickness of histological sections were prepared by a microtome (Yamato Rom 380, Tokyo, Japan) and stained with hematoxylin and eosin (HE) (Wako, Osaka, Japan). The HE-stained sections were examined using an optical microscopy (Nikon Eclipse 80i, Nikon, Tokyo, Japan) for histological and histomorphometrical evaluation.
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