Quantification of Calretinin in Somatosensory Cortex

Somatosensory cortex homogenates were collected at P8 and prepared as described previously [34 (link)]. Protein samples were run on SDS-PAGE and transferred to cellulose acetate membranes. After incubation in TBS containing 5‰ Tween 20 and 5% milk for 1 h at room temperature (RT), the membranes were incubated with primary antibody (rabbit anti-Calretinin, Millipore, AB5054, 1:2000) at 4 °C overnight. After washing in 5‰ Tween 20 in TBS for 30 min, the membranes were incubated with secondary antibody (HRP-linked anti-rabbit IgG, Cell Signaling Technology, 7074S, 1:5000) in TBS buffer for 1 h at room temperature, and immunoreactive bands were visualized with an ECL kit (Thermo Scientific). Quantitative analysis was performed with ImageJ software. The intensity of bands was normalized to the intensity of the corresponding β-tubulin band. An unpaired Student’s t-test was used to determine the significance.