Rabbit anti cofilin

Frozen tissue sections were washed with 1× PBS and blocked with 5% Normal Goat Serum (Abcam; ab7481) for 2 h at room temperature. Sections were washed and incubated with primary antibodies overnight at 4 °C. Primary antibodies used were rabbit anti-MISP (Thermo Scientific; PA5-61995), or rabbit anti-Cofilin (Sigma; C8736). Tissue sections were then washed with 1× PBS and incubated with Alexa-Fluor-568 Phalloidin (Invitrogen; A12380), Wheat German Agglutinin (WGA) 405 M (Biotium; 29028-1), and F(ab’)2-goat ant-rabbit IgG Alexa-Fluor-488 (Molecular probes; A11070) for 2 h at room temperature. Tissue sections were washed with 1× PBS and mounted in ProLong Gold (Invitrogen; P36930).

The validation of the 2-DE data was carried out using Western blot analysis. To assure for the reproducibility of the Western blot analysis, at least three biological and experimental replicates were performed. 40 μg proteins were separated by SDS-PAGE and transferred to Hybond ECL nitrocellulose membrane (GE Healthcare). Immunodetection was performed according to Towbin et al. [24 (link)]. Briefly, membranes were blocked in 5% milk for 2 h at room temperature, followed by overnight incubation at 4°C with diluted specific primary antibody. Mouse monoclonal anti-CRABP1 (1:1000) (abcam), rabbit anti-Cofilin (1:1000) (sigma), mouse monoclonal anti-StAR (1:250) (abcam) and mouse monoclonal anti-ß-actin (1:5000) (sigma) were used as primary antibodies. Molecular Probes Alexa Fluor 647 goat anti-mouse IgG antibody or Alexa Fluor 647 goat anti-rabbit IgG (1:2000) were used as secondary antibodies. Before imaging, the blots were dried in the dark. The blot membranes were scanned at 50 μm resolution on a Fuji FLA-5100 scanner (Fuji Photo) with single laser-emitting excitation light at 635 nm.