All the rats were deeply anesthetized with isoflurane gas before given Buprenorphine (0.05 mg/kg) and a lethal dose of Pentobarbital (100 mg/kg). The rats were transcardially perfused with physiological PBS and then a 4% paraformaldehyde in PBS solution (pH 7.4) at 80 mL/min using a peristaltic pump (World Precision Instruments). The brains were submerged in a 4% paraformaldehyde solution for additional fixation before 2% dimethyl sulfoxide and 20% glycerol (DMSO) at 4°C. The rats used for spine analysis (n = 10) were perfused with ice-cold physiological PBS before brain tissue was stored in a 4% paraformaldehyde solution for 1 wk and then in dimethyl sulfoxide (DMSO) until the brain was sectioned. The brains were cut coronally on Leica CM1950 cryostat (Leica Biosystems), with 40 µm thick sections with six series, in which the first series was put directly on Super Frost glass slides for Cresyl violet (Nissl) staining (Sigma-Aldrich), while the other sections were put on DMSO for immunohistochemistry and stored at −20°C. The brains for spine analysis were cut in 100 µm thick sections and stored at 4°C.
Peristaltic pump
Manufactured by World Precision Instruments
A peristaltic pump is a type of positive displacement pump used to transport fluids by the progressive compression of a flexible tube, called the 'race,' within which the fluid flows. The fluid is contained within the tube and is not in contact with the pump mechanism.
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2 protocols using peristaltic pump
1
Tissue Fixation and Sectioning Protocol
2
Quantification of Myelin Proteins in Mouse Spinal Cord
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To quantify protein amounts, 2-month-old mice were intracardially perfused with 1× PBS using a peristaltic pump (World Precision Instruments) for 3 min. Then, the spinal cord was removed and dissected to isolate the lumbosacral ventrolateral white matter, which was subsequently flash-frozen on dry ice. The tissue was lysed with fresh SDS lysis buffer with Mini Complete protease inhibitor and PhoStop phosphatase inhibitor (Roche) and homogenized with Precellys 24. A final protein concentration of 1 mg ml−1 was prepared in Laemmli buffer and 10 µg of protein was loaded per slot. The proteins were separated on a 12% Bis-Tris mini protein gel and run for 10 min at 80 V and 45 min at 200 V. Subsequently, proteins were transferred onto a PVDF membrane and detected using antibodies (MOG: Abcam, ab109746, rabbit, 1:2,000; secondary HRP-anti-rabbit, Cell Signaling, 7074 S, 1:15,000; MBP: Sigma, Atlas, AMAB91064, mouse, 1:5,000; secondary HRP-anti-mouse, Cell Signaling, 7076, 1:5,000; ACTB: Sigma, A5441, mouse, 1:2,500; secondary HRP-anti-mouse, Cell Signaling, 7076, 1:5,000). The antibodies were incubated separately and subsequently stripped using glycin, SDS and Tween for 10 min before proceeding with the next antibody. Detection was performed using ECL.
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