Computational protocol: Cross-Modal Plasticity Results in Increased Inhibition in Primary Auditory Cortical Areas

Similar protocols

Protocol publication

[…] Animals were euthanized with sodium pentobarbital (>100 mg/kg, IP). Brains were extracted and immediately frozen in 2-methylbutane on dry ice. The brains were stored at −80°C until use. The Western blotting protocol was modified from a previous study []. The auditory cortex surrounded by suprasylvian sulcus was separated and homogenized in 500 μL lysis buffer (10 mM EGTA, 5 mM EDTA, 1 mM DTT in 10 mM PB, pH 7.0, a protease inhibitor cocktail, and 1 mM phenylmethylsulfonyl fluoride (PMSF)). The homogenates were fractionated by centrifuging at 16,000 ×g for 10 min at 4°C. The supernatants were collected as a crude soluble fraction (for GAD Western blots) and placed on ice. The pellets were resuspended in 300 μL 2 mM HEPES, pH 7.2 and ultracentrifuged at 4°C for 45 min at 200,000 ×g. The supernatants from this second spin were discarded. The pellets were resuspended in 280 μL 0.5 mM HEPES, 0.32 M Sucrose pH 7.3 and centrifuged at 4°C for 8 min at 450 ×g. The supernatants were collected as a membrane fraction (for GABA receptor Western blots). Protein concentrations were measured by a plate reader (Spectramax 340PC, Molecular Devices Corporation, Sunnyvale, CA) using a standard BCA assay (BCA Protein assay kit, Thermo Scientific, Rockfold, IL). 10 μg samples per lane were mixed with an equal volume of Laemmli buffer (Bio-Rad, Hercules, CA) and 5% mercaptoethanol, and were denatured at 60°C for 15 min. Samples and standards (Precision Plus protein standard, Bio-Rad) were run on an 8% polyacrylamide SDS gel at 110 V for 100 min. After electrophoresis, proteins were transferred to nitrocellulose membranes using electroblotting (Bio-Rad) at 340 mA for 90 min on ice. The membranes were then stained with Ponceau S and washed in TBST (0.05 M Tris, 0.9% NaCl, 0.1% Tween-20). Non-specific binding was blocked by incubating in 5% non-fat dry milk and 0.1% TBST for 30 min. Blots were incubated overnight with the primary antibodies in 0.1% TBST with 2% non-fat dry milk (rabbit anti-GAD65/67 1 : 10000, Millipore, Billerica, AB1511; rabbit anti- GABAA α1 subunit 1 : 1000, AB5946, Millipore, Billerica, MA; rabbit anti- GABAA γ2, 832-GG2C, 1 : 1000, PhosphoSolutions, Aurora, CO; rabbit anti-NR2A 1 : 2000, AB1555, Millipore, Billerica, MA; rabbit anti-NR2B 1 : 500, AB1557P, Millipore, Billerica, MA). Blots were washed in TBST and incubated with secondary antibodies in 0.1% TBST with 2% non-fat dry milk for 1 hr (horseradish peroxidase-conjugated goat anti-rabbit, 1 : 10,000, Bio-Rad, Hercules, CA). Blots were washed in TBST and TBS and reacted with chemiluminescent reagents (SuperSignal West Pico Chemiluminescent Substrate, ThermoScientific, Rockford, IL). Images were taken with a chemiluminescent ImageQuant LAS4000 Mini (GE Healthcare Life Science, Pittsburgh, PA). The membranes were washed with TBST and stripped with stripping buffer (Restore, ThermoScientific, Rockford, IL). The second immunostaining was the same as described previously except that the primary antibodies were anti-β-actin (from mouse, A2228, 1 : 2000 Sigma-Aldrich, Inc., St. Louis, MO, as a loading control for GAD staining) and anti-pan-cadherin (from mouse, C1821, 1 : 1000, Sigma-Aldrich, Inc., St. Louis, MO, as a loading control for receptor staining). The secondary antibody was horseradish peroxidase-conjugated goat anti-mouse (1 : 10000, Bio-Rad, Hercules, CA). The images were analyzed with ImageJ software (NIH, Bethesda, MA). The optical densities of the GAD and GABA receptor bands were normalized to those of the loading control bands (β-actin and cadherin) from the same membrane. The relative expression level of each protein from the experimental group was then normalized to the average value of the normal group. [...] Statistical comparisons were performed using Sigmastat software (Systat Software Inc., Chicago, IL) and PASW statistic 18 (SPSS Inc., Chicago, IL) and plotted with SigmaPlot software (Systat Software Inc., Chicago, IL) or Matlab (MathWorks Inc. Natick, MA). For within group comparison, paired t-tests were used for normally distributed data, whereas Wilcoxon rank-sum tests were used for non-normally distributed data. For between group comparisons, Student's t-tests were used for normally distributed data, and Mann-Whitney U tests were used for non-normally distributed data. Means are given with standard errors of the mean (±SEM) throughout. […]

Pipeline specifications

Software tools ImageJ, SPSS, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Mustela putorius furo
Diseases Brain Diseases, Cerebrovascular Trauma