The whole-cell patch clamp recordings were conducted as previously described (Paz et al., 2010 (link)). Coronal slices (300–400 m) were cut with a VT1200 vibrotome (Leica, Germany) and removed to a chamber which was superfused with oxygenated artificial cerebrospinal fluid. Whole-cell patch clamp recordings were performed in the layer 5/6 neurons of the M2 region verified with BX51WI microscope (Olympus, Japan) with infrared differential interference contrast (IR-DIC) illumination. The electrophysiological signals were fed into a computer through a Digidata-1440A interface (Axon Instruments, USA) for data acquisition (pClamp 10.0, Axon Instruments, USA). Neurons were held at a membrane potential of −70 mV and characterized by injection of rectangular voltage pulse (5 mV, 50 ms) to monitor the whole-cell membrane capacitance, series resistance and membrane resistance. Neurons were excluded from the study if the series resistance changed by more than 15%. Miniature excitatory postsynaptic current (mEPSCs) were recorded with 1 M TTX and 100 M picrotoxin pre-added to the normal ACSF for at least 15 minutes. Data analyses were conducted using Mini-analysis software (version 6.0, Synaptosoft, USA). Experimenter was blinded to genotype.
Vt1200 vibrotome
Manufactured by Leica camera
Sourced in Germany
The VT1200 vibrotome is a piece of lab equipment designed for sectioning biological samples. It utilizes a vibrating blade to produce thin, uniform sections of tissue for various research and analysis purposes. The core function of the VT1200 is to facilitate the preparation of high-quality tissue sections.
Automatically generated - may contain errors
Lab products found in correlation
Membrane inserts, by Corning (2 mentions)
Laminin, by Merck Group (2 mentions)
Withpoly l orthinine, by Merck Group (2 mentions)
Basal medium eagle, by Thermo Fisher Scientific (2 mentions)
Sp8 confocal microscope, by Leica camera (2 mentions)
Digidata 1440a, by Molecular Devices (1 mentions)
Pclamp 10, by Molecular Devices (1 mentions)
Bx51wi microscope, by Olympus (1 mentions)
3 protocols using vt1200 vibrotome
1
Whole-Cell Patch Clamp Recording in Cortical Neurons
2
Organotypic Forebrain Slice Culture Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Organotypic cultures of mouse coronal forebrain slices were prepared
following published methods45 (link)with some modifications. Whole brains from E14–E18 mouse embryos were
embedded in 4% low-melting point agarose and slices were cut at
250–300 μm using a Leica VT1200 vibrotome in complete HBSS (100
ml of 10× HBSS without Ca or Mg, 2.5 ml of 1M HEPES buffer at pH 7.4, 30
ml of 1M D-glucose, 10 ml of 100 mM CaCl2, 10 ml of 100 mM MgSO4, and 4 ml of 1
M NaHCO3). Slices with visible forebrain structures were placed in membrane
inserts (diameter, 13 mm; pore size, 8 μm; Costar) coated with
Poly-L-orthinine and Laminin (Sigma) overnight. They were cultured in a Basal
Medium Eagle (39 mL, Life Technologies, #21010046) supplemented with
12.9 ml of complete HBSS, 1.35 ml of 1M D-glucose, 250 μl of 200 mM
GlutaMax (Life Technologies) and 5% heat-inactivated horse serum (Life
Technologies, 26050070). Slices were imaged using a Leica SP8 confocal
microscope. Approval for rodent experiments was obtained from the Stanford
University’s Administrative Panel on Laboratory Animal Care (APLAC).
following published methods45 (link)with some modifications. Whole brains from E14–E18 mouse embryos were
embedded in 4% low-melting point agarose and slices were cut at
250–300 μm using a Leica VT1200 vibrotome in complete HBSS (100
ml of 10× HBSS without Ca or Mg, 2.5 ml of 1M HEPES buffer at pH 7.4, 30
ml of 1M D-glucose, 10 ml of 100 mM CaCl2, 10 ml of 100 mM MgSO4, and 4 ml of 1
M NaHCO3). Slices with visible forebrain structures were placed in membrane
inserts (diameter, 13 mm; pore size, 8 μm; Costar) coated with
Poly-L-orthinine and Laminin (Sigma) overnight. They were cultured in a Basal
Medium Eagle (39 mL, Life Technologies, #21010046) supplemented with
12.9 ml of complete HBSS, 1.35 ml of 1M D-glucose, 250 μl of 200 mM
GlutaMax (Life Technologies) and 5% heat-inactivated horse serum (Life
Technologies, 26050070). Slices were imaged using a Leica SP8 confocal
microscope. Approval for rodent experiments was obtained from the Stanford
University’s Administrative Panel on Laboratory Animal Care (APLAC).
3
Organotypic Forebrain Slice Culture Protocol
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!