Computational protocol: Convulsive seizures from experimental focal cortical dysplasia occur independently of cell misplacement

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Protocol publication

[…] Mice were deeply anaesthetised with pentobarbital (50 mg kg−1) and perfused transcardially with ice-cold PBS (pH 7.4) followed by ice-cold paraformaldehyde (PFA, 4%). Perfused brains were also drop fixed in 4% PFA for an additional hour after removal from the skull. Fixed brains were cryoprotected with 30% sucrose in PBS overnight at 4 °C and serially sectioned into 50 μm thick sections, using a freezing microtome. Sections were blocked for 1 h at room temperature in blocking buffer consisting of 2% BSA and 0.3% Triton X-100 in PBS. Floating sections were incubated overnight at 4 °C in primary antibodies diluted in blocking buffer. Following three washes in PBS and an additional 15 min in blocking solution at room temperature, sections were then incubated with Alexa Fluor-tagged secondary antibodies () at 1:1,000 dilution for 2 h at room temperature. ProLong Gold antifade reagent (Life Technologies) is used to mount and preserve stained sections. While blinded to the conditions of the experiment, fluorescence micrograms of serial brain sections were acquired under a fluorescence-enabled stereomicroscope (SZX16, Olympus) at × 0.8 magnification. Stained sections were acquired using a fluorescence confocal microscope (FV1000, Olympus) also in a blind manner. Z-stack images were acquired on the confocal microscope with a × 20 dry objective (N.A. 0.75, Olympus). Images were reconstructed using Imaris 4.0 (Bitplane AG) and Photoshop CS3.Antibodies included anti-: GABA (Sigma, A2052, 1:4,000), GFAP (DAKO, Z0334, 1:4,000), myelin basic protein (Abcam, ab40390, 1:2,000), Nestin (Novus Bio., NM100-1604, 1:300), NeuN (Millipore, MAB377, 1:4,000), NF light chain (Cell Signaling, C28E10, 1:500), pS6 (S240/244, Cell Signaling, D68F8, 1:4,000), SMI 311 (Covance, SMI 311 R, 1:4,000) and Vimentin (MBL, JM-3634-100, 1:500). The host animal is listed in .All analyses were conducted with the analyser knowing only the arbitrarily assigned animal ID (independent of electroporation condition) from which images were taken. Fluorescence intensities and cell sizes were quantified using ImageJ 1.39t (Freeware, Wayne Rasband, NIH). GABA-immunoreactive cells were counted using ImageJ after converting fluorescence micrograms to 8 bit and automatically thresholded. Cells ipsilateral to the side of electroporation were automatically counted with the ‘Analyze Particle' function in ImageJ with the following parameters: minimum cell size 19.6 μm2, circularity 0.40–0.00. […]

Pipeline specifications

Software tools Imaris, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Abnormalities, Drug-Induced, Central Nervous System Diseases, Epilepsy, Malformations of Cortical Development, Leukoencephalopathies
Chemicals Sirolimus